Rnai constructs for inhibiting pnpla3 expression and methods of use thereof

ABSTRACT

The present invention relates to RNAi constructs for reducing expression of the PNPLA3 gene. Methods of using such RNAi constructs to treat or prevent liver disease, nonalcoholic fatty liver disease (NAFLD) are also described.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/597,841, filed Dec. 12, 2017, the contents of which are incorporated herein by reference.

SEQUENCE LISTING

The present application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 12, 2018, is named A-2219-WO-PCT_SL.txt and is 708,961 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for modulating liver expression of patatin-like phospholipase domain-containing 3 (PNPLA3), In particular, the present invention relates to nucleic acid-based therapeutics for reducing PNPLA3 expression via RNA interference and methods of using such nucleic acid-based therapeutics to treat or prevent liver disease, such as nonalcoholic fatty liver disease (NAFLD).

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 12, 2018, is named A-2219-WO-PCT_SL.txt and is 708,961 bytes in size.

BACKGROUND OF THE INVENTION

Comprising a spectrum of hepatic pathologies, nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the world, the prevalence of which doubled in the last 20 years and now is estimated to affect approximately 20% of the world population (Sattar et al. (2014) BMJ 349:g4596; Loomba and Sanyal (2013) Nature Reviews Gastroenterology & hepatology 10(11):686-690; Kim and Kim (2017) Clin Gastroenterol Hepatol 15(4):474-485; Petta et al. (2016) Dig Liver Dis 48(3):333-342). NAFLD begins with the accumulation of triglyceride in the liver and is defined by the presence of cytoplasmic lipid droplets in more than 5% of hepatocytes in an individual 1) without a history of significant alcohol consumption and 2) in which the diagnosis of other types of liver disease have been excluded (Zhu et al (2016) World J Gastroenterol 22(36):8226-33; Rinella (2015) JAMA 313(22):2263-73; Yki-Jarvinen (2016) Diabetologia 59(6):1104-11). In some individuals the accumulation of ectopic fat in the liver, called steatosis, triggers inflammation and hepatocellular injury leading to a more advanced stage of disease called, nonalcoholic steatohepatitis (NASH) (Rinella, supra). As of 2015, 75-100 million Americans are predicted to have NAFLD; NASH accounting for approximately 10-30% of NAFLD diagnoses (Rinella, supra; Younossi et al (2016) Hepatology 64(5): 1577-1586).

Patatin-like phospholipase domain-containing 3 (PNPLA3), formerly known as adiponutrin (ADPN) and calcium-independent phospholipase A2-epsilon (iPLA(2)ε), is a type II transmembrane protein (Wilson et al (2006) J Lipid Res 47(9):1940-9; Jenkins et al (2004) J Biol Chem 279(47):48968-75). Initially identified in adipose cells as a membrane-associated, adipose-enriched protein induced during adipogenesis in mice, it is now well characterized to be expressed in other tissues, including the liver (Wilson et al, supra; Baulande et al (2001) J Biol Chem 276(36):33336-44; Moldes et al. (2006) Eur J Endocrinol 155(3):461-8; Faraj et al. (2006) J Endocrinol 191(2):427-35; Liu et al (2004) J Clin Endocrinol Metab 89(6):2684-9; Lake et al (2005) J Lipid Res 46(11):2477-87). In cell-free biochemical systems, recombinant PNPLA3 protein can exhibit either triacylglycerol lipase or transacylation activity (Jenkins et al., supra; Kumari et al (2012) Cell Metab 15(5):691-702; He et al (2010) J Biol Chem 285(9):6706-15). In hepatocytes, PNPLA3 is expressed on the endoplasmic reticulum and lipid membranes and predominantly exhibits triacylglycerol hydrolase activity (He et al., supra; Huang et al (2010) Proc Natl Acad Sci USA 107(17):7892-7; Ruhanen et al (2014) J Lipid Res 55(4):739-46; Pingitore et al. (2014) Biochim Biophys Acta 1841(4):574-80). Although lacking a secretory signal, data indicates PNPLA3 is secreted and can be found in human plasma as disulfide-bond dependent multimers (Winberg et al. (2014) Biochem Biophys Res Commun 446(4):1114-9). Accordingly, novel therapeutics targeting PNPLA3 function represents a novel approach to reducing PNPLA3 levels and treating hepatologic diseases, such as nonalcoholic fatty liver disease.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the design and generation of RNAi constructs that target the PNPLA3 gene and reduce expression of PNPLA3 in liver cells. The sequence specific inhibition of PNPLA3 expression is useful for treating or preventing conditions associated with PNPLA3 expression, such as liver-related diseases, such as, for example, simple fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis (irreversible, advanced scarring of the liver), or PNPLA3 related obesity. Accordingly, in one embodiment, the present invention provides an RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence that is complementary to a PNPLA3 mRNA sequence. In certain embodiments, the antisense strand comprises a region having at least 15 contiguous nucleotides from an antisense sequence listed in Table 1 or Table 2. In some embodiments, the RNAi of the present invention selectively inhibits PNPLA3-rs738409, PNPLA3-rs738408, and/or PNPLA3-rs738409-rs738408 minor alleles over the reference allele which does not contain these changes.

In some embodiments, the sense strand of the RNAi constructs described herein comprises a sequence that is sufficiently complementary to the sequence of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length. In these and other embodiments, the sense and antisense strands each are about 15 to about 30 nucleotides in length. In some embodiments, the RNAi constructs comprise at least one blunt end. In other embodiments, the RNAi constructs comprise at least one nucleotide overhang. Such nucleotide overhangs may comprise at least 1 to 6 unpaired nucleotides and can be located at the 3′ end of the sense strand, the 3′ end of the antisense strand, or the 3′ end of both the sense and antisense strand. In certain embodiments, the RNAi constructs comprise an overhang of two unpaired nucleotides at the 3′ end of the sense strand and the 3′ end of the antisense strand. In other embodiments, the RNAi constructs comprise an overhang of two unpaired nucleotides at the 3′ end of the antisense strand and a blunt end of the 3′ end of the sense strand/5′ end of the antisense strand.

The RNAi constructs of the invention may comprise one or more modified nucleotides, including nucleotides having modifications to the ribose ring, nucleobase, or phosphodiester backbone. In some embodiments, the RNAi constructs comprise one or more 2′-modified nucleotides. Such 2′-modified nucleotides can include 2′-fluoro modified nucleotides, 2′-O-methyl modified nucleotides, 2′-O-methoxyethyl modified nucleotides, 2′-O-allyl modified nucleotides, bicyclic nucleic acids (BNA), glycol nucleic acids (GNAs), inverted bases (e.g. inverted adenosine) or combinations thereof. In one particular embodiment, the RNAi constructs comprise one or more 2′-fluoro modified nucleotides, 2′-O-methyl modified nucleotides, or combinations thereof. In some embodiments, all of the nucleotides in the sense and antisense strand of the RNAi construct are modified nucleotides.

In some embodiments, the RNAi constructs comprise at least one backbone modification, such as a modified internucleotide or internucleoside linkage. In certain embodiments, the RNAi constructs described herein comprise at least one phosphorothioate internucleotide linkage. In particular embodiments, the phosphorothioate internucleotide linkages may be positioned at the 3′ or 5′ ends of the sense and/or antisense strands.

In some embodiments, the antisense strand and/or the sense strand of the RNAi constructs of the invention may comprise or consist of a sequence from the antisense and sense sequences listed in Tables 1 or 2. In certain embodiments, the RNAi construct may be any one of the duplex compounds listed in any one of Tables 1 to 2.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-D show screening of five siRNA molecules for both dose-dependent mRNA knockdown and functional durability in vivo.

FIG. 2A-G shows effect of PNPLA3 siRNA molecules in vivo in mice, liver weight, confirmation of human PNPLA3 expression, hepatic triglyceride content, serum TIMP1 levels, and histological indication of steatosis or inflammation.

FIG. 3A-G shows effect of PNPLA3 siRNA molecules in vivo, liver weight, confirmation of human PNPLA3 expression, hepatic triglyceride content, serum TIMP1 levels, and histological indication of steatosis or inflammation.

FIG. 4A-D shows the ability of a PNPLA3^(rs738409-rs738408)-specific siRNA molecule to rescue disease-associated phenotypes due to overexpression of PNPLA3^(rs738409-rs738408), hepatic triglyceride content, serum TIMP1 levels, and histological indication of steatosis or inflammation.

FIG. 5A-L shows the ability of a PNPLA3^(rs738409-rs738408)-specific siRNA molecule to prevent the development of early fibrosis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions and methods for regulating the expression of the Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene. In some embodiments, the gene may be within a cell or subject, such as a mammal (e.g. a human). In some embodiments, compositions of the invention comprise RNAi constructs that target a PNPLA3 mRNA and reduce PNPLA3 expression in a cell or mammal. Such RNAi constructs are useful for treating or preventing various forms of liver-related diseases, such as, for example, simple fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis (irreversible, advanced scarring of the liver), or PNPLA3 related obesity.

In 2008, a genome wide association study (GWAS) exploring nonsynomonous sequence variations, or single nucleotide polymorphisms (SNPs), associated with NAFLD identified a variant in PNPLA3, (rs738409[G], encoding I148M; which can be referred to as PNPLA3-rs738409, PNPLA3-ma or PNPLA3-minor allele), as significantly associated with hepatic fat content. Since this initial report, subsequent GWAS confirm PNPLA3 rs738409 as the major genetic determinant of NAFLD, significantly associated with 1) increased levels of the serum biomarker for liver damage, alanine transaminase (ALT), 2) NAFLD incidence, progression and severity, 3) both obese and lean individuals, and 4) the only known SNP shown to be significantly associated with all stages of NAFLD: steatosis, NASH, cirrhosis and hepatic cell carcinoma. The consensus among numerous GWAS indicate the association of PNPLA3 rs738409 with NAFLD is independent of age, gender, ethnicity, metabolic syndrome, body mass index, insulin resistance, and serum lipids. Furthermore, statistical analyses from multiple sources estimate approximately 50% of NAFLD patients carry the PNPLA3 rs738409 mutation. Patients can be homozygous or heterozygous for the PNPLA3 rs738409 mutation. Additionally, it has been discovered that patients having the PNPLA3 rs738409 mutation often also carry an rs738408 mutation 3 base pairs away (Tian et al (2010) Nature Genetics 42:21-23). Thus, a patient can have a PNPLA3-rs738409 minor allele, PNPLA3-rs738408 minor allele or PNPLA3-rs738409-rs738408 double minor allele mutation (PNPLA3-dma).

Investigators have developed mouse models for exploring PNPLA3 function in vivo. To date, no detectable metabolic phenotype has been identified as the result of Pnpla3-deficiency or Pnpla3 over-expression. In contrast, expression of Pnpla3^(I148M) in both transgenic mice and knock-in mice, led to increased hepatic triglyceride levels akin to NAFLD Thus, combined, the in vivo mouse model data points to expression of the mutant Pnpla3^(I148M) protein, and not over-expression of the wild type protein, as the driver of the disease phenotype. These findings, in addition to the high frequency of the minor allele in NAFLD-affected individuals and prevailing association with the disease, underline PNPLA3 rs738409 as a prime therapeutic target for NAFLD.

RNA interference (RNAi) is the process of introducing exogeneous RNA into a cell leading to specific degradation of the mRNA encoding the targeted protein with a resultant decrease in protein expression. Advances in both the RNAi technology and hepatic delivery and growing positive outcomes with other RNAi-based therapies, suggest RNAi as a compelling means to therapeutically treat NAFLD by directly targeting PNPLA3I148M. Numerous GWAS indicate a dose-dependent effect of PNPLA3 rs738409 on NAFLD incidence, progression and severity (GWAS); the odds ratio tending to be double, if not more, for homozygote carriers versus heterozygote carriers, yet still at least two-fold more for heterozygotes versus wild type individuals. Thus, silencing PNPLA3 employing allelic discrimination specificity could be both a potential means to reduce hepatic triglyceride in PNPLA3I148M carriers, but also present a scenario in which heterozygotes may gain benefit without silencing of the wild type allele. Along these lines, we identified SNP-specific short interfering RNAs (siRNA) to PNPLA3I148M and demonstrate proof of concept in vitro. Using both hepatoma cell lines, Hep3B (homozygous for the reference allele, PNPLA3I148I) and HEPG2 (homozygous for the minor allele, PNPLA3I148M), we identified siRNA sequences capable of inhibiting specifically PNPLA3I148M gene expression. The inhibitory effect of these sequences were confirmed by screening on Chinese hamster ovary (CHO) cells over-expressing either PNPLA31148I or PNPLA3I148M. Using adeno-associated virus (AAV) to overexpress human PNPLA3I148M in vivo, we then demonstrated treatment with minor allele-specific SNPs not only specifically reduced human PNPLA3I148M expression in mice, but also significantly reversed hepatic triglyceride accumulation induced by over-expression of human PNPLA3I148M.

As used herein, the term “RNAi construct” refers to an agent comprising a RNA molecule that is capable of downregulating expression of a target gene (e.g. PNPLA3) via a RNA interference mechanism when introduced into a cell. RNA interference is the process by which a nucleic acid molecule induces the cleavage and degradation of a target RNA molecule (e.g. messenger RNA or mRNA molecule) in a sequence-specific manner, e.g. through a RNA induced silencing complex (RISC) pathway. In some embodiments, the RNAi construct comprises a double-stranded RNA molecule comprising two antiparallel strands of contiguous nucleotides that are sufficiently complementary to each other to hybridize to form a duplex region. “Hybridize” or “hybridization” refers to the pairing of complementary polynucleotides, typically via hydrogen bonding (e.g. Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary bases in the two polynucleotides. The strand comprising a region having a sequence that is substantially complementary to a target sequence (e.g. target mRNA) is referred to as the “antisense strand.” The “sense strand” refers to the strand that includes a region that is substantially complementary to a region of the antisense strand. In some embodiments, the sense strand may comprise a region that has a sequence that is substantially identical to the target sequence.

In some embodiments, the invention is an RNAi directed to PNPLA3. In some embodiments, the invention is an RNAi that binds at the PNPLA3 rs738409 site. In some embodiments, the invention is an RNAi that binds at the PNPLA3 rs738408 site. In some embodiments, the invention is an RNAi that binds at both the PNPLA3 rs738409 rs738408 sites. In some embodiments, the invention is an RNAi that preferentially binds PNPLA3 rs738409 over the native PNPLA3 sequence (PNPLA3-ref). In some embodiments, the invention is an RNAi that preferentially binds PNPLA3 rs738408 over the PNPLA3-ref sequence. In some embodiments, the invention is an RNAi that preferentially binds PNPLA3-dma over PNPLA3-ma. In some embodiments, the invention in an RNAi molecule that contains any of the sequences found in Table 1 or 2.

A double-stranded RNA molecule may include chemical modifications to ribonucleotides, including modifications to the ribose sugar, base, or backbone components of the ribonucleotides, such as those described herein or known in the art. Any such modifications, as used in a double-stranded RNA molecule (e.g. siRNA, shRNA, or the like), are encompassed by the term “double-stranded RNA” for the purposes of this disclosure.

As used herein, a first sequence is “complementary” to a second sequence if a polynucleotide comprising the first sequence can hybridize to a polynucleotide comprising the second sequence to form a duplex region under certain conditions, such as physiological conditions. Other such conditions can include moderate or stringent hybridization conditions, which are known to those of skill in the art. A first sequence is considered to be fully complementary (100% complementary) to a second sequence if a polynucleotide comprising the first sequence base pairs with a polynucleotide comprising the second sequence over the entire length of one or both nucleotide sequences without any mismatches. A sequence is “substantially complementary” to a target sequence if the sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary to a target sequence. Percent complementarity can be calculated by dividing the number of bases in a first sequence that are complementary to bases at corresponding positions in a second or target sequence by the total length of the first sequence. A sequence may also be said to be substantially complementary to another sequence if there are no more than 5, 4, 3, 2, or 1 mismatches over a 30 base pair duplex region when the two sequences are hybridized. Generally, if any nucleotide overhangs, as defined herein, are present, the sequence of such overhangs is not considered in determining the degree of complementarity between two sequences. By way of example, a sense strand of 21 nucleotides in length and an antisense strand of 21 nucleotides in length that hybridize to form a 19 base pair duplex region with a 2 nucleotide overhang at the 3′ end of each strand would be considered to be fully complementary as the term is used herein.

In some embodiments, a region of the antisense strand comprises a sequence that is fully complementary to a region of the target RNA sequence (e.g. PNPLA3 mRNA). In such embodiments, the sense strand may comprise a sequence that is fully complementary to the sequence of the antisense strand. In other such embodiments, the sense strand may comprise a sequence that is substantially complementary to the sequence of the antisense strand, e.g. having 1, 2, 3, 4, or 5 mismatches in the duplex region formed by the sense and antisense strands. In certain embodiments, it is preferred that any mismatches occur within the terminal regions (e.g. within 6, 5, 4, 3, 2, or 1 nucleotides of the 5′ and/or 3′ ends of the strands). In one embodiment, any mismatches in the duplex region formed from the sense and antisense strands occur within 6, 5, 4, 3, 2, or 1 nucleotides of the 5′ end of the antisense strand.

In certain embodiments, the sense strand and antisense strand of the double-stranded RNA may be two separate molecules that hybridize to form a duplex region, but are otherwise unconnected. Such double-stranded RNA molecules formed from two separate strands are referred to as “small interfering RNAs” or “short interfering RNAs” (siRNAs). Thus, in some embodiments, the RNAi constructs of the invention comprise a siRNA.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3 ‘-end of one strand and the 5’-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs in the duplex is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.

In other embodiments, the sense strand and the antisense strand that hybridize to form a duplex region may be part of a single RNA molecule, i.e. the sense and antisense strands are part of a self-complementary region of a single RNA molecule. In such cases, a single RNA molecule comprises a duplex region (also referred to as a stem region) and a loop region. The 3′ end of the sense strand is connected to the 5′ end of the antisense strand by a contiguous sequence of unpaired nucleotides, which will form the loop region. The loop region is typically of a sufficient length to allow the RNA molecule to fold back on itself such that the antisense strand can base pair with the sense strand to form the duplex or stem region. The loop region can comprise from about 3 to about 25, from about 5 to about 15, or from about 8 to about 12 unpaired nucleotides. Such RNA molecules with at least partially self-complementary regions are referred to as “short hairpin RNAs” (shRNAs). In some embodiments, the loop region can comprise at least 1, 2, 3, 4, 5, 10, 20, or 25 unpaired nucleotides. In some embodiments, the loop region can have 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer unpaired nucleotides. In certain embodiments, the RNAi constructs of the invention comprise a shRNA. The length of a single, at least partially self-complementary RNA molecule can be from about 35 nucleotides to about 100 nucleotides, from about 45 nucleotides to about 85 nucleotides, or from about 50 to about 60 nucleotides and comprise a duplex region and loop region each having the lengths recited herein.

In some embodiments, the RNAi constructs of the invention comprise a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence that is substantially or fully complementary to a PNPLA3 messenger RNA (mRNA) sequence. As used herein, a “PNPLA3 mRNA sequence” refers to any messenger RNA sequence, including splice variants, encoding a PNPLA3 protein, including PNPLA3 protein variants or isoforms from any species (e.g. mouse, rat, non-human primate, human). PNPLA3 protein is also known as adiponutrin (ADPN) and calcium-independent phospholipase A2-epsilon (iPLA(2)□)).

A PNPLA3 mRNA sequence also includes the transcript sequence expressed as its complementary DNA (cDNA) sequence. A cDNA sequence refers to the sequence of an mRNA transcript expressed as DNA bases (e.g. guanine, adenine, thymine, and cytosine) rather than RNA bases (e.g. guanine, adenine, uracil, and cytosine). Thus, the antisense strand of the RNAi constructs of the invention may comprise a region having a sequence that is substantially or fully complementary to a target PNPLA3 mRNA sequence or PNPLA3 cDNA sequence. A PNPLA3 mRNA or cDNA sequence can include, but is not limited to, any PNPLA3 mRNA or cDNA sequence such as can be derived from the NCBI Reference sequence NM_025225.2.

A region of the antisense strand can be substantially complementary or fully complementary to at least 15 consecutive nucleotides of the PNPLA3 mRNA sequence. In some embodiments, the target region of the PNPLA3 mRNA sequence to which the antisense strand comprises a region of complementarity can range from about 15 to about 30 consecutive nucleotides, from about 16 to about 28 consecutive nucleotides, from about 18 to about 26 consecutive nucleotides, from about 17 to about 24 consecutive nucleotides, from about 19 to about 25 consecutive nucleotides, from about 19 to about 23 consecutive nucleotides, or from about 19 to about 21 consecutive nucleotides. In certain embodiments, the region of the antisense strand comprising a sequence that is substantially or fully complementary to a PNPLA3 mRNA sequence may, in some embodiments, comprise at least 15 contiguous nucleotides from an antisense sequence listed in Table 1 or Table 2. In other embodiments, the antisense sequence comprises at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides from an antisense sequence listed in Table 1 or Table 2. In some embodiments, the sense and/or antisense sequence comprises at least 15 nucleotides from a sequence listed in Table 1 or 2 with no more than 1, 2, or 3 nucleotide mismatches.

The sense strand of the RNAi construct typically comprises a sequence that is sufficiently complementary to the sequence of the antisense strand such that the two strands hybridize under physiological conditions to form a duplex region. A “duplex region” refers to the region in two complementary or substantially complementary polynucleotides that form base pairs with one another, either by Watson-Crick base pairing or other hydrogen bonding interaction, to create a duplex between the two polynucleotides. The duplex region of the RNAi construct should be of sufficient length to allow the RNAi construct to enter the RNA interference pathway, e.g. by engaging the Dicer enzyme and/or the RISC complex. For instance, in some embodiments, the duplex region is about 15 to about 30 base pairs in length. Other lengths for the duplex region within this range are also suitable, such as about 15 to about 28 base pairs, about 15 to about 26 base pairs, about 15 to about 24 base pairs, about 15 to about 22 base pairs, about 17 to about 28 base pairs, about 17 to about 26 base pairs, about 17 to about 24 base pairs, about 17 to about 23 base pairs, about 17 to about 21 base pairs, about 19 to about 25 base pairs, about 19 to about 23 base pairs, or about 19 to about 21 base pairs. In one embodiment, the duplex region is about 17 to about 24 base pairs in length. In another embodiment, the duplex region is about 19 to about 21 base pairs in length.

In some embodiments, an RNAi agent of the invention contains a duplex region of about 24 to about 30 nucleotides that interacts with a target RNA sequence, e.g., an PNPLA3 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells can be broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, 15 processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target 20 mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15: 188).

For embodiments in which the sense strand and antisense strand are two separate molecules (e.g. RNAi construct comprises a siRNA), the sense strand and antisense strand need not be the same length as the length of the duplex region. For instance, one or both strands maybe longer than the duplex region and have one or more unpaired nucleotides or mismatches flanking the duplex region. Thus, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that extend beyond the duplex region at the terminal ends of the strands. Nucleotide overhangs are typically created when the 3′ end of one strand extends beyond the 5′ end of the other strand or when the 5′ end of one strand extends beyond the 3′ end of the other strand. The length of a nucleotide overhang is generally between 1 and 6 nucleotides, land 5 nucleotides, 1 and 4 nucleotides, 1 and 3 nucleotides, 2 and 6 nucleotides, 2 and 5 nucleotides, or 2 and 4 nucleotides. In some embodiments, the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In one particular embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. The nucleotides in the overhang can be ribonucleotides, deoxyribonucleotides, or modified nucleotides as described herein. In some embodiments, the overhang comprises a 5′-uridineuridine-3′ (5′-UU-3′) dinucleotide. In such embodiments, the UU dinucleotide may comprise ribonucleotides or modified nucleotides, e.g. 2′-modified nucleotides. In other embodiments, the overhang comprises a 5′-deoxythymidine-deoxythymidine-3′ (5′-dTdT-3′) dinucleotide.

The nucleotide overhang can be at the 5′ end or 3′ end of one or both strands. For example, in one embodiment, the RNAi construct comprises a nucleotide overhang at the 5′ end and the 3′ end of the antisense strand. In another embodiment, the RNAi construct comprises a nucleotide overhang at the 5′ end and the 3′ end of the sense strand. In some embodiments, the RNAi construct comprises a nucleotide overhang at the 5′ end of the sense strand and the 5′ end of the antisense strand. In other embodiments, the RNAi construct comprises a nucleotide overhang at the 3′ end of the sense strand and the 3′ end of the antisense strand.

The RNAi constructs may comprise a single nucleotide overhang at one end of the double-stranded RNA molecule and a blunt end at the other. A “blunt end” means that the sense strand and antisense strand are fully base-paired at the end of the molecule and there are no unpaired nucleotides that extend beyond the duplex region. In some embodiments, the RNAi construct comprises a nucleotide overhang at the 3′ end of the sense strand and a blunt end at the 5′ end of the sense strand and 3′ end of the antisense strand. In other embodiments, the RNAi construct comprises a nucleotide overhang at the 3′ end of the antisense strand and a blunt end at the 5′ end of the antisense strand and the 3′ end of the sense strand. In certain embodiments, the RNAi construct comprises a blunt end at both ends of the double-stranded RNA molecule. In such embodiments, the sense strand and antisense strand have the same length and the duplex region is the same length as the sense and antisense strands (i.e. the molecule is double-stranded over its entire length).

The sense strand and antisense strand can each independently be about 15 to about 30 nucleotides in length, about 18 to about 28 nucleotides in length, about 19 to about 27 nucleotides in length, about 19 to about 25 nucleotides in length, about 19 to about 23 nucleotides in length, about 21 to about 25 nucleotides in length, or about 21 to about 23 nucleotides in length. In certain embodiments, the sense strand and antisense strand are each about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. In some embodiments, the sense strand and antisense strand have the same length but form a duplex region that is shorter than the strands such that the RNAi construct has two nucleotide overhangs. For instance, in one embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand that are each 21 nucleotides in length, (ii) a duplex region that is 19 base pairs in length, and (iii) nucleotide overhangs of 2 unpaired nucleotides at both the 3′ end of the sense strand and the 3′ end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand that are each 23 nucleotides in length, (ii) a duplex region that is 21 base pairs in length, and (iii) nucleotide overhangs of 2 unpaired nucleotides at both the 3′ end of the sense strand and the 3′ end of the antisense strand. In other embodiments, the sense strand and antisense strand have the same length and form a duplex region over their entire length such that there are no nucleotide overhangs on either end of the double-stranded molecule. In one such embodiment, the RNAi construct is blunt ended and comprises (i) a sense strand and an antisense strand, each of which is 21 nucleotides in length, and (ii) a duplex region that is 21 base pairs in length. In another such embodiment, the RNAi construct is blunt ended and comprises (i) a sense strand and an antisense strand, each of which is 23 nucleotides in length, and (ii) a duplex region that is 23 base pairs in length.

In other embodiments, the sense strand or the antisense strand is longer than the other strand and the two strands form a duplex region having a length equal to that of the shorter strand such that the RNAi construct comprises at least one nucleotide overhang. For example, in one embodiment, the RNAi construct comprises (i) a sense strand that is 19 nucleotides in length, (ii) an antisense strand that is 21 nucleotides in length, (iii) a duplex region of 19 base pairs in length, and (iv) a single nucleotide overhang of 2 unpaired nucleotides at the 3′ end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand that is 21 nucleotides in length, (ii) an antisense strand that is 23 nucleotides in length, (iii) a duplex region of 21 base pairs in length, and (iv) a single nucleotide overhang of 2 unpaired nucleotides at the 3′ end of the antisense strand.

The antisense strand of the RNAi constructs of the invention can comprise the sequence of any one of the antisense sequences listed in Table 1 or Table 2 or the sequence of nucleotides 1-19 of any of these antisense sequences. Each of the antisense sequences listed in Tables 1 and 6 comprises a sequence of 19 consecutive nucleotides (first 19 nucleotides counting from the 5′ end) that is complementary to a PNPLA3 mRNA sequence plus a two nucleotide overhang sequence. Thus, in some embodiments, the antisense strand comprises a sequence of nucleotides 1-19 of any one of SEQ ID NOs: 1-166 or 167-332.

Modified Nucleotides

The RNAi constructs of the invention may comprise one or more modified nucleotides. A “modified nucleotide” refers to a nucleotide that has one or more chemical modifications to the nucleoside, nucleobase, pentose ring, or phosphate group. As used herein, modified nucleotides do not encompass ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate, and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. However, the RNAi constructs may comprise combinations of modified nucleotides, ribonucleotides, and deoxyribonucleotides. Incorporation of modified nucleotides into one or both strands of double-stranded RNA molecules can improve the in vivo stability of the RNA molecules, e.g., by reducing the molecules' susceptibility to nucleases and other degradation processes. The potency of RNAi constructs for reducing expression of the target gene can also be enhanced by incorporation of modified nucleotides.

In certain embodiments, the modified nucleotides have a modification of the ribose sugar. These sugar modifications can include modifications at the 2′ and/or 5′ position of the pentose ring as well as bicyclic sugar modifications. A 2′-modified nucleotide refers to a nucleotide having a pentose ring with a substituent at the 2′ position other than H or OH. Such 2′ modifications include, but are not limited to, 2′-O-alkyl (e.g. O—C1-C10 or O—C1-C10 substituted alkyl), 2′-O-allyl (O—CH2CH═CH2), 2′-C-allyl, 2′-fluoro, 2′-O-methyl (OCH3), 2′-O-methoxyethyl (O—(CH2)2OCH3), 2′-OCF3, 2′-O(CH2)2SCH3, 2′-O-aminoalkyl, 2′-amino (e.g. NH2), 2′-O-ethylamine, and 2′-azido. Modifications at the 5′ position of the pentose ring include, but are not limited to, 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy.

A “bicyclic sugar modification” refers to a modification of the pentose ring where a bridge connects two atoms of the ring to form a second ring resulting in a bicyclic sugar structure. In some embodiments the bicyclic sugar modification comprises a bridge between the 4′ and 2′ carbons of the pentose ring. Nucleotides comprising a sugar moiety with a bicyclic sugar modification are referred to herein as bicyclic nucleic acids or BNAs. Exemplary bicyclic sugar modifications include, but are not limited to, □-L-Methyleneoxy (4′-CH2-O-2′) bicyclicnucleic acid (BNA); □-D-Methyleneoxy (4′-CH2-O-2′) BNA (also referred to as a locked nucleic acid or LNA); Ethyleneoxy (4′-(CH2)2-O-2′) BNA; Aminooxy (4′-CH2-O—N(R)-2′)BNA; Oxyamino (4′-CH2-N(R)—O-2′) BNA; Methyl(methyleneoxy) (4′-CH(CH3)-O-2′) BNA (also referred to as constrained ethyl or cEt); methylene-thio (4′-CH2-S-2′) BNA; methylene-amino (4′-CH2-N(R)-2′) BNA; methyl carbocyclic (4′-CH2-CH(CH3)-2′) BNA; propylene carbocyclic (4′-(CH2)3-2′) BNA; and Methoxy(ethyleneoxy) (4′-CH(CH2OMe)-O-2′) BNA (also referred to as constrained MOE or cMOE). These and other sugar-modified nucleotides that can be incorporated into the RNAi constructs of the invention are described in U.S. Pat. No. 9,181,551, U.S. Patent Publication No. 2016/0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19: 937-954, 2012, all of which are hereby incorporated by reference in their entireties.

In some embodiments, the RNAi constructs comprise one or more 2′-fluoro modified nucleotides, 2′-O-methyl modified nucleotides, 2′-O-methoxyethyl modified nucleotides, 2′-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), or combinations thereof. In certain embodiments, the RNAi constructs comprise one or more 2′-fluoro modified nucleotides, 2′-O-methyl modified nucleotides, 2′-O-methoxyethyl modified nucleotides, or combinations thereof. In one particular embodiment, the RNAi constructs comprise one or more 2′-fluoro modified nucleotides, 2′-O-methyl modified nucleotides or combinations thereof.

Both the sense and antisense strands of the RNAi constructs can comprise one or multiple modified nucleotides. For instance, in some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides. In certain embodiments, all nucleotides in the sense strand are modified nucleotides. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides. In other embodiments, all nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides can be 2′-fluoro modified nucleotides, 2′-O-methyl modified nucleotides, or combinations thereof.

In some embodiments, all pyrimidine nucleotides preceding an adenosine nucleotide in the sense strand, antisense strand, or both strands are modified nucleotides. For example, where the sequence 5′-CA-3′ or 5′-UA-3′ appears in either strand, the cytidine and uridine nucleotides are modified nucleotides, preferably 2′-O-methyl modified nucleotides. In certain embodiments, all pyrimidine nucleotides in the sense strand are modified nucleotides (e.g. 2′-O-methyl modified nucleotides), and the 5′ nucleotide in all occurrences of the sequence 5′-CA-3′ or 5′-UA-3′ in the antisense strand are modified nucleotides (e.g. 2′-O-methyl modified nucleotides). In other embodiments, all nucleotides in the duplex region are modified nucleotides. In such embodiments, the modified nucleotides are preferably 2′-O-methyl modified nucleotides, 2′-fluoro modified nucleotides or combinations thereof.

In embodiments in which the RNAi construct comprises a nucleotide overhang, the nucleotides in the overhang can be ribonucleotides, deoxyribonucleotides, or modified nucleotides. In one embodiment, the nucleotides in the overhang are deoxyribonucleotides, e.g., deoxythymidine. In another embodiment, the nucleotides in the overhang are modified nucleotides. For instance, in some embodiments, the nucleotides in the overhang are 2′-O-methyl modified nucleotides, 2′-fluoro modified nucleotides, 2′-methoxyethyl modified nucleotides, or combinations thereof.

The RNAi constructs of the invention may also comprise one or more modified internucleotide linkages. As used herein, the term “modified internucleotide linkage” refers to an internucleotide linkage other than the natural 3′ to 5′ phosphodiester linkage. In some embodiments, the modified internucleotide linkage is a phosphorous-containing internucleotide linkage, such as a phosphotriester, aminoalkyl phosphotriester, an alkylphosphonate (e.g. methylphosphonate, 3′-alkylene phosphonate), a phosphinate, a phosphoramidate (e.g. 3′-aminophosphoramidate and aminoalkylphosphoramidate), a phosphorothioate (P═S), a chiralphosphorothioate, a phosphorodithioate, a thionophosphoramidate, a thionoalkylphosphonate, athionoalkylphosphotriester, and a boranophosphate. In one embodiment, a modified internucleotide linkage is a 2′ to 5′ phosphodiester linkage. In other embodiments, the modified internucleotide linkage is a non-phosphorous-containing internucleotide linkage and thus can be referred to as a modified internucleoside linkage. Such non-phosphorous-containing linkages include, but are not limited to, morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane linkages (—O—Si(H)2-O—); sulfide, sulfoxide and sulfone linkages; formacetyl and thioformacetyl linkages; alkene containing backbones; sulfamate backbones; methylenemethylimino (—CH2-N(CH3)-O—CH2-) and methylenehydrazino linkages; sulfonate and sulfonamide linkages; amide linkages; and others having mixed N, O, S and CH2 component parts. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g. aminoethylglycine) to create a peptide nucleic acid or PNA, such as those described in U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Other suitable modified internucleotide and internucleoside linkages that may be employed in the RNAi constructs of the invention are described in U.S. Pat. Nos. 6,693,187, 9,181,551, U.S. Patent Publication No. 2016/0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19: 937-954, 2012, all of which are hereby incorporated by reference in their entireties.

In certain embodiments, the RNAi constructs comprise one or more phosphorothioate internucleotide linkages. The phosphorothioate internucleotide linkages may be present in the sense strand, antisense strand, or both strands of the RNAi constructs. For instance, in some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. In other embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. In still other embodiments, both strands comprise 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. The RNAi constructs can comprise one or more phosphorothioate internucleotide linkages at the 3′-end, the 5′-end, or both the 3′- and 5′-ends of the sense strand, the antisense strand, or both strands. For instance, in certain embodiments, the RNAi construct comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 3′-end of the sense strand, the antisense strand, or both strands. In other embodiments, the RNAi construct comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 5′-end of the sense strand, the antisense strand, or both strands. In one embodiment, the RNAi construct comprises a single phosphorothioate internucleotide linkage at the 3′ end of the sense strand and a single phosphorothioate internucleotide linkage at the 3′ end of the antisense strand. In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at the 3′ end of the antisense strand (i.e. a phosphorothioate internucleotide linkage at the first and second internucleotide linkages at the 3′ end of the antisense strand). In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3′ and 5′ ends of the antisense strand. In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3′ and 5′ ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages at the 5′ end of the sense strand. In still another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3′ and 5′ ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages at both the 3′ and 5′ ends of the sense strand (i.e. a phosphorothioate internucleotide linkage at the first and second internucleotide linkages at both the 5′ and 3′ ends of the antisense strand and a phosphorothioate internucleotide linkage at the first and second internucleotide linkages at both the 5′ and 3′ ends of the sense strand). In any of the embodiments in which one or both strands comprises one or more phosphorothioate internucleotide linkages, the remaining internucleotide linkages within the strands can be the natural 3′ to 5′ phosphodiester linkages. For instance, in some embodiments, each internucleotide linkage of the sense and antisense strands is selected from phosphodiester and phosphorothioate, wherein at least one internucleotide linkage is a phosphorothioate.

In embodiments in which the RNAi construct comprises a nucleotide overhang, two or more of the unpaired nucleotides in the overhang can be connected by a phosphorothioate internucleotide linkage. In certain embodiments, all the unpaired nucleotides in a nucleotide overhang at the 3′ end of the antisense strand and/or the sense strand are connected by phosphorothioate internucleotide linkages. In other embodiments, all the unpaired nucleotides in a nucleotide overhang at the 5′ end of the antisense strand and/or the sense strand are connected by phosphorothioate internucleotide linkages. In still other embodiments, all the unpaired nucleotides in any nucleotide overhang are connected by phosphorothioate internucleotide linkages.

In certain embodiments, the modified nucleotides incorporated into one or both of the strands of the RNAi constructs of the invention have a modification of the nucleobase (also referred to herein as “base”). A “modified nucleobase” or “modified base” refers to a base other than the naturally occurring purine bases adenine (A) and guanine (G) and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases can be synthetic or naturally occurring modifications and include, but are not limited to, universal bases, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine (X), hypoxanthine (I), 2-aminoadenine, 6-methyladenine, 6-methylguanine, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine.

In some embodiments, the modified base is a universal base. A “universal base” refers to a base analog that indiscriminately forms base pairs with all of the natural bases in RNA and DNA without altering the double helical structure of the resulting duplex region. Universal bases are known to those of skill in the art and include, but are not limited to, inosine, C-phenyl, C-naphthyl and other aromatic derivatives, azole carboxamides, and nitroazole derivatives, such as 3-nitropyrrol e, 4-nitroindole, 5-nitroindole, and 6-nitroindole.

Other suitable modified bases that can be incorporated into the RNAi constructs of the invention include those described in Herdewijn, Antisense Nucleic Acid Drug Dev., Vol. 10:297-310, 2000 and Peacock et al., J. Org. Chem., Vol. 76: 7295-7300, 2011, both of which are hereby incorporated by reference in their entireties. The skilled person is well aware that guanine, cytosine, adenine, thymine, and uracil may be replaced by other nucleobases, such as the modified nucleobases described above, without substantially altering the base pairing properties of a polynucleotide comprising a nucleotide bearing such replacement nucleobase.

In some embodiments of the RNAi constructs of the invention, the 5′ end of the sense strand, antisense strand, or both the antisense and sense strands comprises a phosphate moiety. As used herein, the term “phosphate moiety” refers to a terminal phosphate group that includes unmodified phosphates (—O—P═O)(OH)OH) as well as modified phosphates. Modified phosphates include phosphates in which one or more of the O and OH groups is replaced with H, O, S, N(R) or alkyl where R is H, an amino protecting group or unsubstituted or substituted alkyl. Exemplary phosphate moieties include, but are not limited to, 5′-monophosphate; 5′diphosphate; 5′-triphosphate; 5′-guanosine cap (7-methylated or non-methylated); 5′-adenosinecap or any other modified or unmodified nucleotide cap structure; 5′-monothiophosphate (phosphorothioate); 5′-monodithiophosphate (phosphorodithioate); 5′-alpha-thiotriphosphate; 5′-gamma-thiotriphosphate, 5′-phosphoramidates; 5′-vinylphosphates; 5′-alkylphosphonates (e.g., alkyl=methyl, ethyl, isopropyl, propyl, etc.); and 5′-alkyletherphosphonates (e.g., alkylether=methoxymethyl, ethoxymethyl, etc.).

The modified nucleotides that can be incorporated into the RNAi constructs of the invention may have more than one chemical modification described herein. For instance, the modified nucleotide may have a modification to the ribose sugar as well as a modification to the nucleobase. By way of example, a modified nucleotide may comprise a 2′ sugar modification (e.g. 2′-fluoro or 2′-methyl) and comprise a modified base (e.g. 5-methyl cytosine or pseudouracil). In other embodiments, the modified nucleotide may comprise a sugar modification in combination with a modification to the 5′ phosphate that would create a modified internucleotide or internucleoside linkage when the modified nucleotide was incorporated into a polynucleotide. For instance, in some embodiments, the modified nucleotide may comprise a sugar modification, such as a 2′-fluoro modification, a 2′-O-methyl modification, or a bicyclic sugar modification, as well as a 5′ phosphorothioate group. Accordingly, in some embodiments, one or both strands of the RNAi constructs of the invention comprise a combination of 2′ modified nucleotides or BNAs and phosphorothioate internucleotide linkages. In certain embodiments, both the sense and antisense strands of the RNAi constructs of the invention comprise a combination of 2′-fluoro modified nucleotides, 2′-O-methyl modified nucleotides, and phosphorothioate internucleotide linkages. Exemplary RNAi constructs comprising modified nucleotides and internucleotide linkages are shown in Table 2.

Function of RNAi Constructs

Preferably, the RNAi constructs of the invention reduce or inhibit the expression of PNPLA3 in cells, particularly liver cells. Accordingly, in one embodiment, the present invention provides a method of reducing PNPLA3 expression in a cell by contacting the cell with any RNAi construct described herein. The cell may be in vitro or in vivo. PNPLA3 expression can be assessed by measuring the amount or level of PNPLA3 mRNA, PNPLA3 protein, or another biomarker linked to PNPLA3 expression. The reduction of PNPLA3 expression in cells or animals treated with an RNAi construct of the invention can be determined relative to the PNPLA3 expression in cells or animals not treated with the RNAi construct or treated with a control RNAi construct. For instance, in some embodiments, reduction of PNPLA3 expression is assessed by (a) measuring the amount or level of PNPLA3 mRNA in liver cells treated with a RNAi construct of the invention, (b) measuring the amount or level of PNPLA3 mRNA in liver cells treated with a control RNAi construct (e.g., RNAi agent directed to a RNA molecule not expressed in liver cells or a RNAi construct having a nonsense or scrambled sequence) or no construct, and (c) comparing the measured PNPLA3 mRNA levels from treated cells in (a) to the measured PNPLA3 mRNA levels from control cells in (b). The PNPLA3 mRNA levels in the treated cells and controls cells can be normalized to RNA levels for a control gene (e.g. 18S ribosomal RNA) prior to comparison. PNPLA3 mRNA levels can be measured by a variety of methods, including Northern blot analysis, nuclease protection assays, fluorescence in situ hybridization (FISH), reverse-transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, and the like.

In other embodiments, reduction of PNPLA3 expression is assessed by (a) measuring the amount or level of PNPLA3 protein in liver cells treated with a RNAi construct of the invention, (b) measuring the amount or level of PNPLA3 protein in liver cells treated with a control RNAi construct (e.g. RNAi agent directed to a RNA molecule not expressed in liver cells or a RNAi construct having a nonsense or scrambled sequence) or no construct, and (c) comparing the measured PNPLA3 protein levels from treated cells in (a) to the measured PNPLA3 protein levels from control cells in (b). Methods of measuring PNPLA3 protein levels are known to those of skill in the art, and include Western Blots, immunoassays (e.g. ELISA), and flow cytometry. An exemplary immunoassay-based method for assessing PNPLA3 protein expression is described in Example 2. Example 3 describes an exemplary method for measuring PNPLA3 mRNA using RNA FISH. Any method capable of measuring PNPLA3 mRNA or protein can be used to assess the efficacy of the RNAi constructs of the invention.

In some embodiments, the methods to assess PNPLA3 expression levels are performed in vitro in cells that natively express PNPLA3 (e.g. liver cells) or cells that have been engineered to express PNPLA3. In certain embodiments, the methods are performed in vitro in liver cells. Suitable liver cells include, but are not limited to, primary hepatocytes (e.g. human, non-human primate, or rodent hepatocytes), HepAD38 cells, HuH-6 cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2 cells. In one embodiment, the liver cells are Hep3B cells. In another embodiment, the liver cells are HepG2 cells.

In other embodiments, the methods to assess PNPLA3 expression levels are performed in vivo. The RNAi constructs and any control RNAi constructs can be administered to an animal (e.g. rodent or non-human primate) and PNPLA3 mRNA or protein levels assessed in liver tissue harvested from the animal following treatment. Alternatively or additionally, a biomarker or functional phenotype associated with PNPLA3 expression can be assessed in the treated animals.

In certain embodiments, expression of PNPLA3 is reduced in liver cells by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by an RNAi construct of the invention. In some embodiments, expression of PNPLA3 is reduced in liver cells by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% by an RNAi construct of the invention. In other embodiments, the expression of PNPLA3 is reduced in liver cells by about 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more by an RNAi construct of the invention. The percent reduction of PNPLA3 expression can be measured by any of the methods described herein as well as others known in the art. For instance, in certain embodiments, the RNAi constructs of the invention inhibit at least 45% of PNPLA3 expression at 5 nM in Hep3B cells (contains wild type PNPLA3) in vitro. In related embodiments, the RNAi constructs of the invention inhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of PNPLA3 expression at 5 nM in Hep3B cells in vitro. In other embodiments, the RNAi constructs of the invention inhibit at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% of PNPLA3 expression at 5 nM in Hep3B cells in vitro. In certain embodiments, the RNAi constructs of the invention inhibit at least 45% of PNPLA3 expression at 5 nM in HepG2 cells (contains the PNPLA3-rs738409-rs738408 double minor allele) in vitro. In related embodiments, the RNAi constructs of the invention inhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of PNPLA3 expression at 5 nM in HepG2 cells in vitro. In other embodiments, the RNAi constructs of the invention inhibit at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% of PNPLA3 expression at 5 nM in HepG2 cells in vitro. In certain embodiments, the RNAi constructs of the invention inhibit at least 45% of PNPLA3 expression at 5 nM in CHO transfected cells expressing human PNPLA3 I1481 or I148M cells in vitro. In related embodiments, the RNAi constructs of the invention inhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of PNPLA3 expression at 5 nM in CHO transfected cells expressing human PNPLA3 I1481 or I148M in vitro. In other embodiments, the RNAi constructs of the invention inhibit at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% of PNPLA3 expression at 5 nM in CHO transfected cells expressing human PNPLA3 I1481 or I148M in vitro. Reduction of PNPLA3 can be measured using a variety of techniques including RNA FISH or droplet digital PCR, as described in Examples 2 and 3.

In some embodiments, an IC50 value is calculated to assess the potency of an RNAi construct of the invention for inhibiting PNPLA3 expression in liver cells. An “IC50 value” is the dose/concentration required to achieve 50% inhibition of a biological or biochemical function. The IC50 value of any particular substance or antagonist can be determined by constructing a dose-response curve and examining the effect of different concentrations of the substance or antagonist on expression levels or functional activity in any assay. IC50 values can be calculated for a given antagonist or substance by determining the concentration needed to inhibit half of the maximum biological response or native expression levels. Thus, the IC50 value for any RNAi construct can be calculated by determining the concentration of the RNAi construct needed to inhibit half of the native PNPLA3 expression level in liver cells (e.g. PNPLA3 expression level in control liver cells) in any assay, such as the immunoassay or RNA FISH assay or droplet digital PCR assays described in the Examples. The RNAi constructs of the invention may inhibit PNPLA3 expression in liver cells (e.g. Hep3B cells) with an IC50 of less than about 20 nM. For example, the RNAi constructs inhibit PNPLA3 expression in liver cells with an IC50 of about 0.001 nM to about 20 nM, about 0.001 nM to about 10 nM, about 0.001 nM to about 5 nM, about 0.001 nM to about 1 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, or about 0.1 nM to about 1 nM. In certain embodiments, the RNAi construct inhibits PNPLA3 expression in liver cells (e.g. Hep3B cells) with an IC50 of about 1 nM to about 10 nM. The RNAi constructs of the invention may inhibit PNPLA3 expression in liver cells (e.g. HepG2 cells) with an IC50 of less than about 20 nM. For example, the RNAi constructs inhibit PNPLA3 expression in liver cells with an IC50 of about 0.001 nM to about 20 nM, about 0.001 nM to about 10 nM, about 0.001 nM to about 5 nM, about 0.001 nM to about 1 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, or about 0.1 nM to about 1 nM. In certain embodiments, the RNAi construct inhibits PNPLA3 expression in liver cells (e.g. HepG2 cells) with an IC50 of about 1 nM to about 10 nM. The RNAi constructs of the invention may inhibit PNPLA3 expression in liver cells (e.g. CHO transfected cells expressing human PNPLA3 I1481 or I148M) with an IC50 of less than about 20 nM. For example, the RNAi constructs inhibit PNPLA3 expression in liver cells with an IC50 of about 0.001 nM to about 20 nM, about 0.001 nM to about 10 nM, about 0.001 nM to about 5 nM, about 0.001 nM to about 1 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, or about 0.1 nM to about 1 nM. In certain embodiments, the RNAi construct inhibits PNPLA3 expression in liver cells (e.g. CHO transfected cells expressing human PNPLA3 I1481 or I148M) with an IC50 of about 1 nM to about 10 nM.

The RNAi constructs of the invention can readily be made using techniques known in the art, for example, using conventional nucleic acid solid phase synthesis. The polynucleotides of the RNAi constructs can be assembled on a suitable nucleic acid synthesizer utilizing standard nucleotide or nucleoside precursors (e.g. phosphoramidites). Automated nucleic acid synthesizers are sold commercially by several vendors, including DNA/RNA synthesizers from Applied Biosystems (Foster City, Calif.), MerMade synthesizers from BioAutomation (Irving, Tex.), and OligoPilot synthesizers from GE Healthcare Life Sciences (Pittsburgh, Pa.).

The 2′ silyl protecting group can be used in conjunction with acid labile dimethoxytrityl (DMT) at the 5′ position of ribonucleosides to synthesize oligonucleotides via phosphoramidite chemistry. Final deprotection conditions are known not to significantly degrade RNA products. All syntheses can be conducted in any automated or manual synthesizer on large, medium, or small scale. The syntheses may also be carried out in multiple well plates, columns, or glass slides.

The 2′-O-silyl group can be removed via exposure to fluoride ions, which can include any source of fluoride ion, e.g., those salts containing fluoride ion paired with inorganic counterions, e.g., cesium fluoride and potassium fluoride or those salts containing fluoride ion paired with an organic counterion, e.g., a tetraalkylammonium fluoride. A crown ether catalyst can be utilized in combination with the inorganic fluoride in the deprotection reaction. Preferred fluoride ion source are tetrabutylammonium fluoride or aminohydrofluorides (e.g., combining aqueous HF with triethylamine in a dipolar aprotic solvent, e.g., dimethylformamide).

The choice of protecting groups for use on the phosphite triesters and phosphotriesters can alter the stability of the triesters towards fluoride. Methyl protection of the phosphotriester or phosphitetriester can stabilize the linkage against fluoride ions and improve process yields.

Since ribonucleosides have a reactive 2′ hydroxyl substituent, it can be desirable to protect the reactive 2′ position in RNA with a protecting group that is orthogonal to a 5′-O-dimethoxytrityl protecting group, e.g., one stable to treatment with acid. Silyl protecting groups meet this criterion and can be readily removed in a final fluoride deprotection step that can result in minimal RNA degradation.

Tetrazole catalysts can be used in the standard phosphoramidite coupling reaction. Preferred catalysts include, e.g., tetrazole, S-ethyl-tetrazole, benzylthiotetrazole, pnitrophenyltetrazole.

As can be appreciated by the skilled artisan, further methods of synthesizing the RNAi constructs described herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Other synthetic chemistry transformations, protecting groups (e.g., for hydroxyl, amino, etc. present on the bases) and protecting group methodologies (protection and deprotection) useful in synthesizing the RNAi constructs described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof Custom synthesis of RNAi agents is also available from several commercial vendors, including Dharmacon, Inc. (Lafayette, Colo.), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, Calif.).

The RNAi constructs of the invention may comprise a ligand. As used herein, a “ligand” refers to any compound or molecule that is capable of interacting with another compound or molecule, directly or indirectly. The interaction of a ligand with another compound or molecule may elicit a biological response (e.g. initiate a signal transduction cascade, induce receptor mediated endocytosis) or may just be a physical association. The ligand can modify one or more properties of the double-stranded RNA molecule to which is attached, such as the pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties of the RNA molecule.

The ligand may comprise a serum protein (e.g., human serum albumin, low-density lipoprotein, globulin), a cholesterol moiety, a vitamin (biotin, vitamin E, vitamin B12), a folate moiety, a steroid, a bile acid (e.g. cholic acid), a fatty acid (e.g., palmitic acid, myristic acid), a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), a glycoside, a phospholipid, or antibody or binding fragment thereof (e.g. antibody or binding fragment that targets the RNAi construct to a specific cell type, such as liver). Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-BisO(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., antennapedia peptide, Tat peptide, RGD peptides), alkylating agents, polymers, such as polyethylene glycol (PEG) (e.g., PEG-40K), poly amino acids, and polyamines (e.g. spermine, spermidine).

In certain embodiments, the ligands have endosomolytic properties. The endosomolytic ligands promote the lysis of the endosome and/or transport of the RNAi construct of the invention, or its components, from the endosome to the cytoplasm of the cell. The endosomolytic ligand may be a polycationic peptide or peptidomimetic which shows pH dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand assumes its active conformation at endosomal pH. The “active” conformation is that conformation in which the endosomolytic ligand promotes lysis of the endosome and/or transport of the RNAi construct of the invention, or its components, from the endosome to the cytoplasm of the cell. Exemplary endosomolytic ligands include the GALA peptide (Subbarao et al., Biochemistry, Vol. 26: 2964-2972, 1987), the EALA peptide (Vogel et al., J. Am. Chern. Soc., Vol. 118: 1581-1586, 1996), and their derivatives (Turk et al., Biochem. Biophys. Acta, Vol. 1559: 56-68, 2002). In one embodiment, the endosomolytic component may contain a chemical group (e.g., an amino acid) which will undergo a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

In some embodiments, the ligand comprises a lipid or other hydrophobic molecule. In one embodiment, the ligand comprises a cholesterol moiety or other steroid. Cholesterol conjugated oligonucleotides have been reported to be more active than their unconjugated counterparts (Manoharan, Antisense Nucleic Acid Drug Development, Vol. 12: 103-228, 2002). Ligands comprising cholesterol moieties and other lipids for conjugation to nucleic acid molecules have also been described in U.S. Pat. Nos. 7,851,615; 7,745,608; and 7,833,992, all of which are hereby incorporated by reference in their entireties. In another embodiment, the ligand comprises a folate moiety. Polynucleotides conjugated to folate moieties can be taken up by cells via a receptor-mediated endocytosis pathway. Such folate-polynucleotide conjugates are described in U.S. Pat. No. 8,188,247, which is hereby incorporated by reference in its entirety.

Given that PNPLA3 is expressed in liver cells (e.g. hepatocytes), in certain embodiments, it is desirable to specifically deliver the RNAi construct to those liver cells. In some embodiments, RNAi constructs can be specifically targeted to the liver by employing ligands that bind to or interact with proteins expressed on the surface of liver cells. For example, in certain embodiments, the ligands may comprise antigen binding proteins (e.g. antibodies or binding fragments thereof (e.g. Fab, scFv)) that specifically bind to a receptor expressed on hepatocytes.

In certain embodiments, the ligand comprises a carbohydrate. A “carbohydrate” refers to a compound made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Carbohydrates include, but are not limited to, the sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides, such as starches, glycogen, cellulose and polysaccharide gums. In some embodiments, the carbohydrate incorporated into the ligand is a monosaccharide selected from a pentose, hexose, or heptose and di- and tri-saccharides including such monosaccharide units. In other embodiments, the carbohydrate incorporated into the ligand is an amino sugar, such as galactosamine, glucosamine, N-acetylgalactosamine, and N-acetylglucosamine.

In some embodiments, the ligand comprises a hexose or hexosamine. The hexose may be selected from glucose, galactose, mannose, fucose, or fructose. The hexosamine may be selected from fructosamine, galactosamine, glucosamine, or mannosamine. In certain embodiments, the ligand comprises glucose, galactose, galactosamine, or glucosamine. In one embodiment, the ligand comprises glucose, glucosamine, or N-acetylglucosamine. In another embodiment, the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine. In particular embodiments, the ligand comprises N-acetyl-galactosamine. Ligands comprising glucose, galactose, and N-acetyl-galactosamine (GalNAc) are particularly effective in targeting compounds to liver cells. See, e.g., D'Souza and Devarajan, J. Control Release, Vol. 203: 126-139, 2015. Examples of GalNAc- or galactose-containing ligands that can be incorporated into the RNAi constructs of the invention are described in U.S. Pat. Nos. 7,491,805; 8,106,022; and 8,877,917; U.S. Patent Publication No. 20030130186; and WIPO Publication No. WO2013166155, all of which are hereby incorporated by reference in their entireties.

In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, a “multivalent carbohydrate moiety” refers to a moiety comprising two or more carbohydrate units capable of independently binding or interacting with other molecules. For example, a multivalent carbohydrate moiety comprises two or more binding domains comprised of carbohydrates that can bind to two or more different molecules or two or more different sites on the same molecule. The valency of the carbohydrate moiety denotes the number of individual binding domains within the carbohydrate moiety. For instance, the terms “monovalent,” “bivalent,” “trivalent,” and “tetravalent” with reference to the carbohydrate moiety refer to carbohydrate moieties with one, two, three, and four binding domains, respectively. The multivalent carbohydrate moiety may comprise a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetyl-galactosamine moiety, a multivalent N-acetyl-glucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the ligand comprises a multivalent galactose moiety. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the multivalent carbohydrate moiety is bivalent, trivalent, or tetravalent. In such embodiments, the multivalent carbohydrate moiety can be bi-antennary or tri-antennary. In one particular embodiment, the multivalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another particular embodiment, the multivalent galactose moiety is trivalent or tetravalent. Exemplary trivalent and tetravalent GalNAc-containing ligands for incorporation into the RNAi constructs of the invention are described in detail below.

The ligand can be attached or conjugated to the RNA molecule of the RNAi construct directly or indirectly. For instance, in some embodiments, the ligand is covalently attached directly to the sense or antisense strand of the RNAi construct. In other embodiments, the ligand is covalently attached via a linker to the sense or antisense strand of the RNAi construct. The ligand can be attached to nucleobases, sugar moieties, or internucleotide linkages of polynucleotides (e.g. sense strand or antisense strand) of the RNAi constructs of the invention. Conjugation or attachment to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms. In certain embodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a ligand. Conjugation or attachment to pyrimidine nucleobases or derivatives thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be attached to a ligand. Conjugation or attachment to sugar moieties of nucleotides can occur at any carbon atom. Example carbon atoms of a sugar moiety that can be attached to a ligand include the 2′, 3′, and 5′ carbon atoms. The 1′ position can also be attached to a ligand, such as in an a basic residue. Internucleotide linkages can also support ligand attachments. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate, phosphoroamidate, and the like), the ligand can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide-containing internucleoside linkages (e.g., PNA), the ligand can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

In certain embodiments, the ligand may be attached to the 3′ or 5′ end of either the sense or antisense strand. In certain embodiments, the ligand is covalently attached to the 5′ end of the sense strand. In other embodiments, the ligand is covalently attached to the 3′ end of the sense strand. For example, in some embodiments, the ligand is attached to the 3′-terminal nucleotide of the sense strand. In certain such embodiments, the ligand is attached at the 3′-position of the 3′-terminal nucleotide of the sense strand. In alternative embodiments, the ligand is attached near the 3′ end of the sense strand, but before one or more terminal nucleotides (i.e. before 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is attached at the 2′-position of the sugar of the 3′-terminal nucleotide of the sense strand.

In certain embodiments, the ligand is attached to the sense or antisense strand via a linker. A “linker” is an atom or group of atoms that covalently joins a ligand to a polynucleotide component of the RNAi construct. The linker may be from about 1 to about 30 atoms in length, from about 2 to about 28 atoms in length, from about 3 to about 26 atoms in length, from about 4 to about 24 atoms in length, from about 6 to about 20 atoms in length, from about 7 to about 20 atoms in length, from about 8 to about 20 atoms in length, from about 8 to about 18 atoms in length, from about 10 to about 18 atoms in length, and from about 12 to about 18 atoms in length. In some embodiments, the linker may comprise a bifunctional linking moiety, which generally comprises an alkyl moiety with two functional groups. One of the functional groups is selected to bind to the compound of interest (e.g. sense or antisense strand of the RNAi construct) and the other is selected to bind essentially any selected group, such as a ligand as described herein. In certain embodiments, the linker comprises a chain structure or an oligomer of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups that are typically employed in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.

Linkers that may be used to attach a ligand to the sense or antisense strand in the RNAi constructs of the invention include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl. Preferred substituent groups for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, the linkers are cleavable. A cleavable linker is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In some embodiments, the cleavable linker is cleaved at least 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or more, or at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linkers are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linker by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linker by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linker may comprise a moiety that is susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable group that is cleaved at a preferred pH, thereby releasing the RNA molecule from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable group that is cleavable by a particular enzyme. The type of cleavable group incorporated into a linker can depend on the cell to be targeted. For example, liver-targeting ligands can be linked to RNA molecules through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other types of cells rich in esterases include cells of the lung, renal cortex, and testis. Linkers that contain peptide bonds can be used when targeting cells rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linker. It will also be desirable to also test the candidate cleavable linker for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In some embodiments, useful candidate linkers are cleaved at least 2, 4, 10, 20, 50, 70, or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

In other embodiments, redox cleavable linkers are utilized. Redox cleavable linkers are cleaved upon reduction or oxidation. An example of reductively cleavable group is a disulfide linking group (—S—S—). To determine if a candidate cleavable linker is a suitable “reductively cleavable linker,” or for example is suitable for use with a particular RNAi construct and particular ligand, one can use one or more methods described herein. For example, a candidate linker can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent known in the art, which mimics the rate of cleavage that would be observed in a cell, e.g., a target cell. The candidate linkers can also be evaluated under conditions which are selected to mimic blood or serum conditions. In a specific embodiment, candidate linkers are cleaved by at most 10% in the blood. In other embodiments, useful candidate linkers are degraded at least 2, 4, 10, 20, 50, 70, or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions).

In yet other embodiments, phosphate-based cleavable linkers are cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that hydrolyzes phosphate groups in cells are enzymes, such as phosphatases in cells. Examples of phosphate-based cleavable groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Specific embodiments include —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —SP(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. Another specific embodiment is —O—P(O)(OH)—O—. These candidate linkers can be evaluated using methods analogous to those described above.

In other embodiments, the linkers may comprise acid cleavable groups, which are groups that are cleaved under acidic conditions. In some embodiments, acid cleavable groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents, such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes, can provide a cleaving environment for acid cleavable groups. Examples of acid cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A specific embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiaryalkyl group such as dimethyl, pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

In other embodiments, the linkers may comprise ester-based cleavable groups, which are cleaved by enzymes, such as esterases and amidases in cells. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. Ester cleavable groups have the general formula —C(O)O—, or —OC(O)—. These candidate linkers can be evaluated using methods analogous to those described above.

In further embodiments, the linkers may comprise peptide-based cleavable groups, which are cleaved by enzymes, such as peptidases and proteases in cells. Peptide-based cleavable groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

Other types of linkers suitable for attaching ligands to the sense or antisense strands in the RNAi constructs of the invention are known in the art and can include the linkers described in U.S. Pat. Nos. 7,723,509; 8,017,762; 8,828,956; 8,877,917; and 9,181,551, all of which are hereby incorporated by reference in their entireties.

In certain embodiments, the ligand covalently attached to the sense or antisense strand of the RNAi constructs of the invention comprises a GalNAc moiety, e.g, a multivalent GalNAc moiety. In some embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 3′ end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 5′ end of the sense strand. In yet other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3′ end of the sense strand. In still other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5′ end of the sense strand.

In some embodiments, the RNAi constructs of the invention may be delivered to a cell or tissue of interest by administering a vector that encodes and controls the intracellular expression of the RNAi construct. A “vector” (also referred to herein as an “expression vector) is a composition of matter which can be used to deliver a nucleic acid of interest to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like. A vector can be replicated in a living cell, or it can be made synthetically.

Generally, a vector for expressing an RNAi construct of the invention will comprise one or more promoters operably linked to sequences encoding the RNAi construct. The phrase “operably linked” or “under transcriptional control” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide sequence to control the initiation of transcription by RNA polymerase and expression of the polynucleotide sequence. A “promoter” refers to a sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene sequence. Suitable promoters include, but are not limited to, RNA pol I, pol II, HI or U6 RNA pol III, and viral promoters (e.g. human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat). In some embodiments, a HI or U6RNA pol III promoter is preferred. The promoter can be a tissue-specific or inducible promoter. Of particular interest are liver-specific promoters, such as promoter sequences from human alpha!-antitrypsin gene, albumin gene, hemopexin gene, and hepatic lipase gene. Inducible promoters include promoters regulated by ecdysone, estrogen, progesterone, tetracycline, and isopropyl-PD1-thiogalactopyranoside (IPTG).

In some embodiments in which the RNAi construct comprises a siRNA, the two separate strands (sense and antisense strand) can be expressed from a single vector or two separate vectors. For example, in one embodiment, the sequence encoding the sense strand is operably linked to a promoter on a first vector and the sequence encoding the antisense strand is operably linked to a promoter on a second vector. In such an embodiment, the first and second vectors are co-introduced, e.g., by infection or transfection, into a target cell, such that the sense and anti sense strands, once transcribed, will hybridize intracellularly to form the siRNA molecule. In another embodiment, the sense and antisense strands are transcribed from two separate promoters located in a single vector. In some such embodiments, the sequence encoding the sense strand is operably linked to a first promoter and the sequence encoding the antisense strand is operably linked to a second promoter, wherein the first and second promoters are located in a single vector. In one embodiment, the vector comprises a first promoter operably linked to a sequence encoding the siRNA molecule, and a second promoter operably linked to the same sequence in the opposite direction, such that transcription of the sequence from the first promoter results in the synthesis of the sense strand of the siRNA molecule and transcription of the sequence from the second promoter results in synthesis of the antisense strand of the siRNA molecule.

In other embodiments in which the RNAi construct comprises a shRNA, a sequence encoding the single, at least partially self-complementary RNA molecule is operably linked to a promoter to produce a single transcript. In some embodiments, the sequence encoding the shRNA comprises an inverted repeat joined by a linker polynucleotide sequence to produce the stem and loop structure of the shRNA following transcription.

In some embodiments, the vector encoding an RNAi construct of the invention is a viral vector. Various viral vector systems that are suitable to express the RNAi constructs described herein include, but are not limited to, adenoviral vectors, retroviral vectors (e.g., lentiviral vectors, maloney murine leukemia virus), adeno-associated viral vectors; herpes simplex viral vectors; SV 40 vectors; polyoma viral vectors; papilloma viral vectors; picornaviral vectors; and pox viral vectors (e.g. vaccinia virus). In certain embodiments, the viral vector is a retroviral vector (e.g. lentiviral vector).

Various vectors suitable for use in the invention, methods for inserting nucleic acid sequences encoding siRNA or shRNA molecules into vectors, and methods of delivering the vectors to the cells of interest are within the skill of those in the art. See, e.g., Dornburg, Gene Therap., Vol. 2: 301-310, 1995; Eglitis, Biotechniques, Vol. 6: 608-614, 1988; Miller, HumGene Therap., Vol. 1: 5-14, 1990; Anderson, Nature, Vol. 392: 25-30, 1998; Rubinson D A et al., Nat. Genet., Vol. 33: 401-406, 2003; Brummelkamp et al., Science, Vol. 296: 550-553, 2002; Brummelkamp et al., Cancer Cell, Vol. 2: 243-247, 2002; Lee et al., Nat Biotechnol, Vol. 20:500-505, 2002; Miyagishi et al., Nat Biotechnol, Vol. 20: 497-500, 2002; Paddison et al., Genes Dev, Vol. 16: 948-958, 2002; Paul et al., Nat Biotechnol, Vol. 20: 505-508, 2002; Sui et al., Proc Natl Acad Sci USA, Vol. 99: 5515-5520, 2002; and Yu et al., Proc Natl Acad Sci USA, Vol. 99:6047-6052, 2002, all of which are hereby incorporated by reference in their entireties.

The present invention also includes pharmaceutical compositions and formulations comprising the RNAi constructs described herein and pharmaceutically acceptable carriers, excipients, or diluents. Such compositions and formulations are useful for reducing expression of PNPLA3 in a subject in need thereof Where clinical applications are contemplated, pharmaceutical compositions and formulations will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier, excipient, or diluent” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the RNAi constructs of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or RNAi constructs of the compositions.

Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, type and extent of disease or disorder to be treated, or dose to be administered. In some embodiments, the pharmaceutical compositions are formulated based on the intended route of delivery. For instance, in certain embodiments, the pharmaceutical compositions are formulated for parenteral delivery. Parenteral forms of delivery include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such an embodiment, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such an embodiment, the pharmaceutical composition may include a targeting ligand (e.g. GalNAc containing ligands described herein).

In some embodiments, the pharmaceutical compositions comprise an effective amount of an RNAi construct described herein. An “effective amount” is an amount sufficient to produce a beneficial or desired clinical result. In some embodiments, an effective amount is an amount sufficient to reduce PNPLA3 expression in hepatocytes of a subject. In some embodiments, an effective amount may be an amount sufficient to only partially reduce PNPLA3 expression, for example, to a level comparable to expression of the wild-type PNPLA3 allele in human heterozygotes. Human heterozygous carriers of loss of function PNPLA3 variant alleles were reported to have lower serum levels of non-HDL cholesterol and a lower risk of coronary artery disease and myocardial infarction as compared to non-carriers (Nioi et al., New England Journal of Medicine, Vol. 374(22):2131-2141, 2016). Thus, without being bound by theory, it is believed that partial reduction of PNPLA3 expression may be sufficient to achieve the beneficial reduction of serum non-HDL cholesterol and reduction of risk of coronary artery disease and myocardial infarction.

An effective amount of an RNAi construct of the invention may be from about 0.01 mg/kg body weight to about 100 mg/kg body weight, about 0.05 mg/kg body weight to about 75 mg/kg body weight, about 0.1 mg/kg body weight to about 50 mg/kg body weight, about 1 mg/kg to about 30 mg/kg body weight, about 2.5 mg/kg of body weight to about 20 mg/kg bodyweight, or about 5 mg/kg body weight to about 15 mg/kg body weight. In certain embodiments, a single effective dose of an RNAi construct of the invention may be about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10 mg/kg. The pharmaceutical composition comprising an effective amount of RNAi construct can be administered weekly, biweekly, monthly, quarterly, or biannually. The precise determination of what would be considered an effective amount and frequency of administration may be based on several factors, including a patient's size, age, and general condition, type of disorder to be treated (e.g. myocardial infarction, heart failure, coronary artery disease, hypercholesterolemia), particular RNAi construct employed, and route of administration. Estimates of effective dosages and in vivo half-lives for any particular RNAi construct of the invention can be ascertained using conventional methods and/or testing in appropriate animal models.

Administration of the pharmaceutical compositions of the present invention may be via any common route so long as the target tissue is available via that route. Such routes include, but are not limited to, parenteral (e.g., subcutaneous, intramuscular, intraperitoneal or intravenous), oral, nasal, buccal, intradermal, transdermal, and sublingual routes, or by direct injection into liver tissue or delivery through the hepatic portal vein. In some embodiments, the pharmaceutical composition is administered parenterally. For instance, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

Colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes, may be used as delivery vehicles for the RNAi constructs of the invention or vectors encoding such constructs. Commercially available fat emulsions that are suitable for delivering the nucleic acids of the invention include Intralipid®, Liposyn®, Liposyn®II, Nutrilipid, and other similar lipid emulsions. A preferred colloidal system for use as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle). The RNAi constructs of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, RNAi constructs of the invention may be complexed to lipids, in particular to cationic lipids. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), and dipalmitoyl phosphatidylcholine (DPPC)), distearolyphosphatidyl choline), negative (e.g., dimyristoylphosphatidyl glycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidyl ethanolamine (DOTMA)). The preparation and use of such colloidal dispersion systems is well known in the art. Exemplary formulations are also disclosed in U.S. Pat. Nos. 5,981,505; 6,217,900; 6,383,512; 5,783,565; 7,202,227; 6,379,965; 6,127,170; 5,837,533; 6,747,014; and WO03/093449.

In some embodiments, the RNAi constructs of the invention are fully encapsulated in a lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a noncationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are exceptionally useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO00/03683. The nucleic acid-lipid particles typically have a mean diameter of about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO96/40964.

The pharmaceutical compositions suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The compositions of the present invention generally may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with free amino groups) derived from inorganic acids (e.g., hydrochloric or phosphoric acids), or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like). Salts formed with the free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like).

For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA standards. In certain embodiments, a pharmaceutical composition of the invention comprises or consists of a sterile saline solution and an RNAi construct described herein. In other embodiments, a pharmaceutical composition of the invention comprises or consists of an RNAi construct described herein and sterile water (e.g. water for injection, WFI). In still other embodiments, a pharmaceutical composition of the invention comprises or consists of an RNAi construct described herein and phosphate-buffered saline (PBS).

In some embodiments, the pharmaceutical compositions of the invention are packaged with or stored within a device for administration. Devices for injectable formulations include, but are not limited to, injection ports, pre-filled syringes, auto injectors, injection pumps, on-body injectors, and injection pens. Devices for aerosolized or powder formulations include, but are not limited to, inhalers, insufflators, aspirators, and the like. Thus, the present invention includes administration devices comprising a pharmaceutical composition of the invention for treating or preventing one or more of the disorders described herein.

Methods for Inhibiting PNPLA3 Expression

The present invention also provides methods of inhibiting expression of a PNPLA3 gene in a cell. The methods include contacting a cell with an RNAi agent, e.g., double stranded RNAi agent, in an amount effective to inhibit expression of PNPLA3 in the cell, thereby inhibiting expression of PNPLA3 in the cell. Contacting of a cell with an RNAi agent, e.g., a double stranded RNAi agent, may be done in vitro or in vivo. Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting a cell are also possible.

The present invention provides methods for reducing or inhibiting expression of PNPLA3 in a subject in need thereof as well as methods of treating or preventing conditions, diseases, or disorders associated with PNPLA3 expression or activity. A “condition, disease, or disorder associated with PNPLA3 expression” refers to conditions, diseases, or disorders in which PNPLA3 expression levels are altered or where elevated expression levels of PNPLA3 are associated with an increased risk of developing the condition, disease or disorder.

Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc3 ligand, or any other ligand that directs the RNAi agent to a site of interest.

In one embodiment, contacting a cell with an RNAi includes “introducing” or “delivering the RNAi into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an RNAi can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an RNAi into a cell may be in vitro and/or in vivo. For example, for in vivo introduction, RNAi can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a PNPLA3” is intended to refer to inhibition of expression of any PNPLA3 gene (such as, e.g., a mouse PNPLA3 gene, a rat PNPLA3 gene, a monkey PNPLA3 gene, or a human PNPLA3 gene) as well as variants or mutants of a PNPLA3 gene. Thus, the PNPLA3 gene may be a wild-type PNPLA3 gene, a mutant PNPLA3 gene (such as a mutant PNPLA3 gene giving rise to amyloid deposition), or a transgenic PNPLA3 gene in the context of a genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of a PNPLA3 gene” includes any level of inhibition of a PNPLA3 gene, e.g., at least partial suppression of the expression of a PNPLA3 gene. The expression of the PNPLA3 gene may be assessed based on the level, or the change in the level, of any variable associated with PNPLA3 gene expression, e.g., PNPLA3 mRNA level, PNPLA3 protein level, or the number or extent of amyloid deposits. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.

Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with PNPLA3 expression compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control). In some embodiments of the methods of the invention, expression of a PNPLA3 gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

Inhibition of the expression of a PNPLA3 gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a PNPLA3 gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with an RNAi agent of the invention, or by administering an RNAi agent of the invention to a subject in which the cells are or were present) such that the expression of a PNPLA3 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s)). In preferred embodiments, the inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells, using the following formula:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, inhibition of the expression of a PNPLA3 gene may be assessed in terms of a reduction of a parameter that is functionally linked to PNPLA3 gene expression, e.g., PNPLA3 protein expression or Hedgehog pathway protein activities. PNPLA3 gene silencing may be determined in any cell expressing PNPLA3, either constitutively or by genomic engineering, and by any assay known in the art.

Inhibition of the expression of a PNPLA3 protein may be manifested by a reduction in the level of the PNPLA3 protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for the assessment of mRNA suppression, the inhibiton of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess the inhibition of the expression of a PNPLA3 gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the invention. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent.

The level of PNPLA3 mRNA that is expressed by a cell or group of cells, or the level of circulating PNPLA3 mRNA, may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of PNPLA3 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the PNPLA3 gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis. Circulating PNPLA3 mRNA may be detected using methods the described in PCT/US2012/043584, the entire contents of which are hereby incorporated herein by reference.

In one embodiment, the level of expression of PNPLA3 is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific PNPLA3. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to PNPLA3 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of PNPLA3 mRNA.

An alternative method for determining the level of expression of PNPLA3 in a sample involves the process of nucleic acid amplification and/or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6: 1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, the level of expression of PNPLA3 is determined by quantitative fluorogenic RT-PCR {i.e., the TaqMan™ System). The expression levels of PNPLA3 mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of PNPLA3 expression level may also comprise using nucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of these methods is described and exemplified in the Examples presented herein.

The level of PNPLA3 protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), Immunoelectrophoresis, Western blotting, radioimmunoassay (MA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the invention can be monitored by detecting or monitoring a reduction in a symptom of a PNPLA3 disease, such as reduction in edema swelling of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitals, prodrome; laryngeal swelling; nonpruritic rash; nausea; vomiting; or abdominal pain. These symptoms may be assessed in vitro or in vivo using any method known in the art.

In some embodiments of the methods of the invention, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. The inhibition of expression of PNPLA3 may be assessed using measurements of the level or change in the level of PNPLA3 mRNA or PNPLA3 protein in a sample derived from fluid or tissue from the specific site within the subject. In preferred embodiments, the site is selected from the group consisting of liver, choroid plexus, retina, and pancreas. The site may also be a subsection or subgroup of cells from any one of the aforementioned sites. The site may also include cells that express a particular type of receptor.

Methods of Treating or Preventing PNPLA3-Associated Diseases

The present invention provides therapeutic and prophylactic methods which include administering to a subject with a PNPLA3-associated disease, disorder, and/or condition, or prone to developing, a PNPLA3-associated disease, disorder, and/or condition, compositions comprising an RNAi agent, or pharmaceutical compositions comprising an RNAi agent, or vectors comprising an RNAi of the invention. Non-limiting examples of PNPLA3-associated diseases include, for example, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). In one embodiment, the PNPLA3-associated disease is NAFLD. In another embodiment, the PNPLA3-associated disease is NASH. In another embodiment, the PNPLA3-associated disease is fatty liver (steatosis). In another embodiment, the PNPLA3-associated disease is insulin resistance. In another embodiment, the PNPLA3-associated disease is not insulin resistance.

In certain embodiments, the present invention provides a method for reducing the expression of PNPLA3 in a patient in need thereof comprising administering to the patient any of the RNAi constructs described herein. The term “patient,” as used herein, refers to a mammal, including humans, and can be used interchangeably with the term “subject.” Preferably, the expression level of PNPLA3 in hepatocytes in the patient is reduced following administration of the RNAi construct as compared to the PNPLA3 expression level in a patient not receiving the RNAi construct.

The methods of the invention are useful for treating a subject having a PNPLA3-associated disease, e.g., a subject that would benefit from reduction in PNPLA3 gene expression and/or PNPLA3 protein production. In one aspect, the present invention provides methods of reducing the level of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) gene expression in a subject having nonalcoholic fatty liver disease (NAFLD). In another aspect, the present invention provides methods of reducing the level of PNPLA3 protein in a subject with NAFLD. The present invention also provides methods of reducing the level of activity of the hedgehog pathway in a subject with NAFLD.

In another aspect, the present invention provides methods of treating a subject having an NAFLD. In one aspect, the present invention provides methods of treating a subject having an PNPLA3-associated disease, e.g., fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). The treatment methods (and uses) of the invention include administering to the subject, e.g., a human, a therapeutically effective amount of an RNAi agent of the invention targeting a PNPLA3 gene or a pharmaceutical composition comprising an RNAi agent of the invention targeting a PNPLA3 gene or a vector of the invention comprising an RNAi agent targeting an PNPLA3 gene.

In one aspect, the invention provides methods of preventing at least one symptom in a subject having NAFLD, e.g., the presence of elevated hedgehog signaling pathways, fatigue, weakness, weight loss, loss of apetite, nausea, abdominal pain, spider-like blood vessels, yellowing of the skin and eyes (jaundice), itching, fluid build up and swelling of the legs (edema), abdomen swelling (ascites), and mental confusion. The methods include administering to the subject a therapeutically effective amount of the RNAi agent, e.g. dsRNA, pharmaceutical compositions, or vectors of the invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduction in PNPLA3 gene expression.

In another aspect, the present invention provides uses of a therapeutically effective amount of an RNAi agent of the invention for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of PNPLA3 gene expression. In a further aspect, the present invention provides uses of an RNAi agent, e.g., a dsRNA, of the invention targeting an PNPLA3 gene or pharmaceutical composition comprising an RNAi agent targeting an PNPLA3 gene in the manufacture of a medicament for treating a subject, e.g., a subject that would benefit from a reduction and/or inhibition of PNPLA3 gene expression and/or PNPLA3 protein production, such as a subject having a disorder that would benefit from reduction in PNPLA3 gene expression, e.g., a PNPLA3-associated disease.

In another aspect, the invention provides uses of an RNAi, e.g., a dsRNA, of the invention for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of PNPLA3 gene expression and/or PNPLA3 protein production.

In a further aspect, the present invention provides uses of an RNAi agent of the invention in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from a reduction and/or inhibition of PNPLA3 gene expression and/or PNPLA3 protein production, such as a PNPLA3-associated disease.

In one embodiment, an RNAi agent targeting PNPLA3 is administered to a subject having a PNPLA3-associated disease, e.g., nonalcoholic fatty liver disease (NAFLD), such that the expression of a PNPLA3 gene, e.g., in a cell, tissue, blood or other tissue or fluid of the subject are reduced by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more when the dsRNA agent is administered to the subject.

The methods and uses of the invention include administering a composition described herein such that expression of the target PNPLA3 gene is decreased, such as for about 1, 2, 3, 4 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours. In one embodiment, expression of the target PNPLA3 gene is decreased for an extended duration, e.g., at least about two, three, four, five, six, seven days or more, e.g., about one week, two weeks, three weeks, or about four weeks or longer.

Administration of the dsRNA according to the methods and uses of the invention may result in a reduction of the severity, signs, symptoms, and/or markers of such diseases or disorders in a patient with a PNPLA3-associated disease, e.g., nonalcoholic fatty liver disease (NAFLD). By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%. Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of NAFLD may be assessed, for example, by periodic monitoring of NAFLD symptoms, liver fat levels, or expression of downstream genes. Comparison of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an RNAi targeting PNPLA3 or pharmaceutical composition thereof, “effective against” an PNPLA3-associated disease indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating NAFLD and/or an PNPLA3-associated disease and the related causes.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given RNAi drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

Subjects can be administered a therapeutic amount of RNAi, such as about 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg, 0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA, 2.8 mg/kg dsRNA, 2.9 mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kg dsRNA, 3.3 mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kg dsRNA, 3.7 mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kg dsRNA, 4.1 mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kg dsRNA, 4.5 mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kg dsRNA, 4.9 mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kg dsRNA, 5.3 mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6 mg/kg dsRNA, 5.7 mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kg dsRNA, 6.1 mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kg dsRNA, 6.5 mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8 mg/kg dsRNA, 6.9 mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2 mg/kg dsRNA, 7.3 mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6 mg/kg dsRNA, 7.7 mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0 mg/kg dsRNA, 8.1 mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg/kg dsRNA, 8.4 mg/kg dsRNA, 8.5 mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8 mg/kg dsRNA, 8.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kg dsRNA, 9.3 mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kg dsRNA, 9.7 mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 10 mg/kg dsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA, 30 mg/kg dsRNA, 35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA, or about 50 mg/kg dsRNA. In one embodiment, subjects can be administered 0.5 mg/kg of the dsRNA. Values and ranges intermediate to the recited values are also intended to be part of this invention.

Administration of the RNAi can reduce the presence of PNPLA3 protein levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

Before administration of a full dose of the RNAi, patients can be administered a smaller dose, such as a 5% infusion, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Owing to the inhibitory effects on PNPLA3 expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.

An RNAi of the invention may be administered in “naked” form, where the modified or unmodified RNAi agent is directly suspended in aqueous or suitable buffer solvent, as a “free RNAi.” A free RNAi is administered in the absence of a pharmaceutical composition. The free RNAi may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolality of the buffer solution containing the RNAi can be adjusted such that it is suitable for administering to a subject.

Alternatively, an RNAi of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of PNPLA3 gene expression are those having nonalcoholic fatty liver disease (NAFLD) and/or an PNPLA3-associated disease or disorder as described herein.

Treatment of a subject that would benefit from a reduction and/or inhibition of PNPLA3 gene expression includes therapeutic and prophylactic treatment.

The invention further provides methods and uses of an RNAi agent or a pharmaceutical composition thereof for treating a subject that would benefit from reduction and/or inhibition of PNPLA3 gene expression, e.g., a subject having a PNPLA3-associated disease, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders.

For example, in certain embodiments, an RNAi targeting a PNPLA3 gene is administered in combination with, e.g., an agent useful in treating an PNPLA3-associated disease as described elsewhere herein. For example, additional therapeutics and therapeutic methods suitable for treating a subject that would benefit from reduction in PNPLA3 expression, e.g., a subject having a PNPLA3-associated disease, include an RNAi agent targeting a different portion of the PNPLA3 gene, a therapeutic agent, and/or procedures for treating a PNPLA3-associated disease or a combination of any of the foregoing.

In certain embodiments, a first RNAi agent targeting a PNPLA3 gene is administered in combination with a second RNAi agent targeting a different portion of the PNPLA3 gene. For example, the first RNAi agent comprises a first sense strand and a first antisense strand forming a double stranded region, wherein substantially all of the nucleotides of said first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, wherein said first sense strand is conjugated to a ligand attached at the 3′-terminus, and wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker; and the second RNAi agent comprises a second sense strand and a second antisense strand forming a double stranded region, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, wherein the second sense strand is conjugated to a ligand attached at the 3′-terminus, and wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, all of the nucleotides of the first and second sense strand and/or all of the nucleotides of the first and second antisense strand comprise a modification.

In one embodiment, the at least one of the modified nucleotides is selected from the group consisting of a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxly-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5 ‘-phosphate, and a nucleotide comprising a 5’-phosphate mimic.

In certain embodiments, a first RNAi agent targeting a PNPLA3 gene is administered in combination with a second RNAi agent targeting a gene that is different from the PNPLA3 gene. For example, the RNAi agent targeting the PNPLA3 gene may be administered in combination with an RNAi agent targeting the SCAP gene. The first RNAi agent targeting a PNPLA3 gene and the second RNAi agent targeting a gene different from the PNPLA3 gene, e.g., the SCAP gene, may be administered as parts of the same pharmaceutical composition. Alternatively, the first RNAi agent targeting a PNPLA3 gene and the second RNAi agent targeting a gene different from the PNPLA3 gene, e.g., the SCAP gene, may be administered as parts of different pharmaceutical compositions.

The RNAi agent and an additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.

The present invention also provides methods of using an RNAi agent of the invention and/or a composition containing an RNAi agent of the invention to reduce and/or inhibit PNPLA3 expression in a cell. In other aspects, the present invention provides an RNAi of the invention and/or a composition comprising an RNAi of the invention for use in reducing and/or inhibiting PNPLA3 gene expression in a cell. In yet other aspects, use of an RNAi of the invention and/or a composition comprising an RNAi of the invention for the manufacture of a medicament for reducing and/or inhibiting PNPLA3 gene expression in a cell are provided. In still other aspects, the present invention provides an RNAi of the invention and/or a composition comprising an RNAi of the invention for use in reducing and/or inhibiting PNPLA3 protein production in a cell. In yet other aspects, use of an RNAi of the invention and/or a composition comprising an RNAi of the invention for the manufacture of a medicament for reducing and/or inhibiting PNPLA3 protein production in a cell are provided. The methods and uses include contacting the cell with an RNAi, e.g., a dsRNA, of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of an PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene or inhibiting PNPLA3 protein production in the cell.

Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of PNPLA3 may be determined by determining the mRNA expression level of PNPLA3 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR, by determining the protein level of PNPLA3 using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques, flow cytometry methods, ELISA, and/or by determining a biological activity of PNPLA3.

In the methods and uses of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may be any cell that expresses an PNPLA3 gene, e.g., a cell from a subject having NAFLD or a cell comprising an expression vector comprising a PNPLA3 gene or portion of a PNPLA3 gene. A cell suitable for use in the methods and uses of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell.

PNPLA3 gene expression may be inhibited in the cell by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

PNPLA3 protein production may be inhibited in the cell by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

The in vivo methods and uses of the invention may include administering to a subject a composition containing an RNAi, where the RNAi includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the PNPLA3 gene of the mammal to be treated. When the organism to be treated is a human, the composition can be administered by any means known in the art including, but not limited to subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection. In one embodiment, the compositions are administered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. A depot injection may release the RNAi in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of PNPLA3, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi to the subject.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods for inhibiting the expression of an PNPLA3 gene in a mammal, e.g., a human. The present invention also provides a composition comprising an RNAi, e.g., a dsRNA, that targets an PNPLA3 gene in a cell of a mammal for use in inhibiting expression of the PNPLA3 gene in the mammal. In another aspect, the present invention provides use of an RNAi, e.g., a dsRNA, that targets an PNPLA3 gene in a cell of a mammal in the manufacture of a medicament for inhibiting expression of the PNPLA3 gene in the mammal.

The methods and uses include administering to the mammal, e.g., a human, a composition comprising an RNAi, e.g., a dsRNA, that targets an PNPLA3 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene in the mammal.

Reduction in gene expression can be assessed in peripheral blood sample of the RNAi-administered subject by any methods known it the art, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g., ELISA or Western blotting, described herein. In one embodiment, a tissue sample serves as the tissue material for monitoring the reduction in PNPLA3 gene and/or protein expression. In another embodiment, a blood sample serves as the tissue material for monitoring the reduction in PNPLA3 gene and/or protein expression.

In one embodiment, verification of RISC medicated cleavage of target in vivo following administration of RNAi agent is done by performing 5′-RACE or modifications of the protocol as known in the art (Lasham A et al., (2010) Nucleic Acid Res., 38 (3) p-e19) (Zimmermann et al. (2006) Nature 441: 111-4).

It is understood that all ribonucleic acid sequences disclosed herein can be converted to deoxyribonucleic acid sequences by substituting a thymine base for a uracil base in the sequence. Likewise, all deoxyribonucleic acid sequences disclosed herein can be converted to ribonucleic acid sequences by substituting a uracil base for a thymine base in the sequence. Deoxyribonucleic acid sequences, ribonucleic acid sequences, and sequences containing mixtures of deoxyribonucleotides and ribonucleotides of all sequences disclosed herein are included in the invention.

Additionally, any nucleic acid sequences disclosed herein may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified polynucleotides is, in certain instances, arbitrary. For example, a polynucleotide comprising a nucleotide having a 2′-OH substituent on the ribose sugar and a thymine base could be described as a DNA molecule having a modified sugar (2′-OH for the natural 2′-H of DNA) or as an RNA molecule having a modified base (thymine (methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of a further example and without limitation, a polynucleotide having the sequence “ATCGATCG” encompasses any polynucleotides having such a sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and polynucleotides having other modified bases, such as “ATmeCGAUCG,” wherein meC indicates a cytosine base comprising a methyl group at the 5-position.

The following examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and are not to be construed as limiting the scope of the appended claims.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention. To the extent that any of the definitions or terms provided in the references incorporated by reference differ from the terms and discussion provided herein, the present terms and definitions control.

EQUIVALENTS

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and describe the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof.

The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.

Example 1: Selection, Design and Synthesis of Modified PNPLA3 siRNA Molecules

The identification and selection of optimal sequences for therapeutic siRNA molecules targeting patatin-like phospholipase domain-containing 3 (PNPLA3) were identified using bioinformatics analysis of a human PNPLA3 transcript (NM_025225.2). Table 1 shows sequences identified as having therapeutic properties. Throughout the various sequences, {INVAB} is an inverted A basic, {INVDA} is an inverted deoxythymidine, GNA is a glycol nucleic acid, dT is deoxythymidine and dC is deoxycytosine.

TABLE 1 siRNA sequences directed to PNPLA3 SEQ ID SEQ ID Duplex NO: NO: No. Sense sequence (5′-3′) (sense) Antisense sequence (5′-3′)  (antisense) D-1000 GGGCAAUAAAGUACCUGCUUU 1 AGCAGGUACUUUAUUGCCCUU 2 D-1001 CGGCCAAUGUCCACCAGCUUU 3 AGCUGGUGGACAUUGGCCGUU 4 D-1002 GGUCCAGCCUGAACUUCUUUU 5 AAGAAGUUCAGGCUGGACCUU 6 D-1003 GCUUCAUCCCCUUCUACAGUU 7 CUGUAGAAGGGGAUGAAGCUU 8 D-1004 GCGGCUUCCUGGGCUUCUAUU 9 UAGAAGCCCAGGAAGCCGCUU 10 D-1005 GCCUCUGAGCUGAGUUGGUUU 11 ACCAACUCAGCUCAGAGGCUU 12 D-1006 GUGACAACGUACCCUUCAUUU 13 AUGAAGGGUACGUUGUCACUU 14 D-1007 CCCGCCUCCAGGUCCCAAAUU 15 UUUGGGACCUGGAGGCGGGUU 16 D-1008 CUUCAUCCCCUUCUACAGUUU 17 ACUGUAGAAGGGGAUGAAGUU 18 D-1009 GGUAUGUUCCUGCUUCAUGUU 19 CAUGAAGCAGGAACAUACCUU 20 D-1010 GUAUGUUCCUGCUUCAUGCUU 21 GCAUGAAGCAGGAACAUACUU 22 D-1011 UAUGUUCCUGCUUCAUGCCUU 23 GGCAUGAAGCAGGAACAUAUU 24 D-1012 AUGUUCCUGCUUCAUGCCCUU 25 GGGCAUGAAGCAGGAACAUUU 26 D-1013 UGUUCCUGCUUCAUGCCCUUU 27 AGGGCAUGAAGCAGGAACAUU 28 D-1014 GUUCCUGCUUCAUGCCCUUUU 29 AAGGGCAUGAAGCAGGAACUU 30 D-1015 UUCCUGCUUCAUGCCCUUCUU 31 GAAGGGCAUGAAGCAGGAAUU 32 D-1016 UCCUGCUUCAUGCCCUUCUUU 33 AGAAGGGCAUGAAGCAGGAUU 34 D-1017 CCUGCUUCAUGCCCUUCUAUU 35 UAGAAGGGCAUGAAGCAGGUU 36 D-1018 CUGCUUCAUGCCCUUCUACUU 37 GUAGAAGGGCAUGAAGCAGUU 38 D-1019 UGCUUCAUGCCCUUCUACAUU 39 UGUAGAAGGGCAUGAAGCAUU 40 D-1020 GCUUCAUGCCCUUCUACAGUU 41 CUGUAGAAGGGCAUGAAGCUU 42 D-1021 CUUCAUGCCCUUCUACAGUUU 43 ACUGUAGAAGGGCAUGAAGUU 44 D-1022 UUCAUGCCCUUCUACAGUGUU 45 CACUGUAGAAGGGCAUGAAUU 46 D-1023 UCAUGCCCUUCUACAGUGGUU 47 CCACUGUAGAAGGGCAUGAUU 48 D-1024 CAUGCCCUUCUACAGUGGCUU 49 GCCACUGUAGAAGGGCAUGUU 50 D-1025 AUGCCCUUCUACAGUGGCCUU 51 GGCCACUGUAGAAGGGCAUUU 52 D-1026 UGCCCUUCUACAGUGGCCUUU 53 AGGCCACUGUAGAAGGGCAUU 54 D-1027 GCCCUUCUACAGUGGCCUUUU 55 AAGGCCACUGUAGAAGGGCUU 56 D-1028 GGUAUGUUCCUGCUUCAUCUU 57 GAUGAAGCAGGAACAUACCUU 58 D-1029 GUAUGUUCCUGCUUCAUCCUU 59 GGAUGAAGCAGGAACAUACUU 60 D-1030 UAUGUUCCUGCUUCAUCCCUU 61 GGGAUGAAGCAGGAACAUAUU 62 D-1031 AUGUUCCUGCUUCAUCCCCUU 63 GGGGAUGAAGCAGGAACAUUU 64 D-1032 UGUUCCUGCUUCAUCCCCUUU 65 AGGGGAUGAAGCAGGAACAUU 66 D-1033 GUUCCUGCUUCAUCCCCUUUU 67 AAGGGGAUGAAGCAGGAACUU 68 D-1034 UUCCUGCUUCAUCCCCUUCUU 69 GAAGGGGAUGAAGCAGGAAUU 70 D-1035 UCCUGCUUCAUCCCCUUCUUU 71 AGAAGGGGAUGAAGCAGGAUU 72 D-1036 CCUGCUUCAUCCCCUUCUAUU 73 UAGAAGGGGAUGAAGCAGGUU 74 D-1037 CUGCUUCAUCCCCUUCUACUU 75 GUAGAAGGGGAUGAAGCAGUU 76 D-1038 UGCUUCAUCCCCUUCUACAUU 77 UGUAGAAGGGGAUGAAGCAUU 78 D-1039 UUCAUCCCCUUCUACAGUGUU 79 CACUGUAGAAGGGGAUGAAUU 80 D-1040 UCAUCCCCUUCUACAGUGGUU 81 CCACUGUAGAAGGGGAUGAUU 82 D-1041 CAUCCCCUUCUACAGUGGCUU 83 GCCACUGUAGAAGGGGAUGUU 84 D-1042 UCCCCUUCUACAGUGGCCUUU 85 AGGCCACUGUAGAAGGGGAUU 86 D-1043 GAUCAGGACCCGAGCCGAUUU 87 AUCGGCUCGGGUCCUGAUCUU 88 D-1044 UGGGCUUCUACCACGUCGUUU 89 ACGACGUGGUAGAAGCCCAUU 90 D-1045 GAGCGAGCACGCCCCGCAUUU 91 AUGCGGGGCGUGCUCGCUCUU 92 D-1046 UGCACUGCGUCGGCGUCCUUU 93 AGGACGCCGACGCAGUGCAUU 94 D-1047 UGGAGCAGACUCUGCAGGUUU 95 ACCUGCAGAGUCUGCUCCAUU 96 D-1048 UGCAGGUCCUCUCAGAUCUUU 97 AGAUCUGAGAGGACCUGCAUU 98 D-1049 CCCGGCCAAUGUCCACCAUUU 99 AUGGUGGACAUUGGCCGGGUU 100 D-1050 UUCUACAGUGGCCUUAUCUUU 101 AGAUAAGGCCACUGUAGAAUU 102 D-1051 UCUACAGUGGCCUUAUCCUUU 103 AGGAUAAGGCCACUGUAGAUU 104 D-1052 CUUCCUUCAGAGGCGUGCUUU 105 AGCACGCCUCUGAAGGAAGUU 106 D-1053 UUCCUUCAGAGGCGUGCGAUU 107 UCGCACGCCUCUGAAGGAAUU 108 D-1054 GCGUGCGAUAUGUGGAUGUUU 109 ACAUCCACAUAUCGCACGCUU 110 D-1055 CGUGCGAUAUGUGGAUGGAUU 111 UCCAUCCACAUAUCGCACGUU 112 D-1056 UGGAUGGAGGAGUGAGUGAUU 113 UCACUCACUCCUCCAUCCAUU 114 D-1057 ACGUACCCUUCAUUGAUGUUU 115 ACAUCAAUGAAGGGUACGUUU 116 D-1058 UGGACAUCACCAAGCUCAUUU 117 AUGAGCUUGGUGAUGUCCAUU 118 D-1059 CACCUGCGUCUCAGCAUCUUU 119 AGAUGCUGAGACGCAGGUGUU 120 D-1060 ACCUGCGUCUCAGCAUCCUUU 121 AGGAUGCUGAGACGCAGGUUU 122 D-1061 CCAGAGACUGGUGACAUGUUU 123 ACAUGUCACCAGUCUCUGGUU 124 D-1062 AUGGCUUCCAGAUAUGCCUUU 125 AGGCAUAUCUGGAAGCCAUUU 126 D-1063 CCGCCUCCAGGUCCCAAAUUU 127 AUUUGGGACCUGGAGGCGGUU 128 D-1064 UACCUGCUGGUGCUGAGGUUU 129 ACCUCAGCACCAGCAGGUAUU 130 D-1065 ACCUGCUGGUGCUGAGGGUUU 131 ACCCUCAGCACCAGCAGGUUU 132 D-1066 CUCUCCACCUUUCCCAGUUUU 133 AACUGGGAAAGGUGGAGAGUU 134 D-1067 UUUUUCACCUAACUAAAAUUU 135 AUUUUAGUUAGGUGAAAAAUU 136 D-1068 CGGCCAAUGUCCACCAGCUUU 137 AGCUGGUGGACAUUGGCCGUU 138 D-1069 GGUCCAGCCUGAACUUCUUUU 139 AAGAAGUUCAGGCUGGACCUU 140 D-1070 GCGGCUUCCUGGGCUUCUAUU 141 UAGAAGCCCAGGAAGCCGCUU 142 D-1071 GUGACAACGUACCCUUCAUUU 143 AUGAAGGGUACGUUGUCACUU 144 D-1072 GGUAUGUUCCUGCUUCAUGUU 145 CAUGAAGCAGGAACAUACCUU 146 D-1073 GUAUGUUCCUGCUUCAUGCUU 147 GCAUGAAGCAGGAACAUACUU 148 D-1074 UGUUCCUGCUUCAUGCCCUUU 149 AGGGCAUGAAGCAGGAACAUU 150 D-1075 GUUCCUGCUUCAUGCCCUUUU 151 AAGGGCAUGAAGCAGGAACUU 152 D-1076 CCUGCUUCAUGCCCUUCUAUU 153 UAGAAGGGCAUGAAGCAGGUU 154 D-1077 GCUUCAUGCCCUUCUACAGUU 155 CUGUAGAAGGGCAUGAAGCUU 156 D-1078 CUUCAUGCCCUUCUACAGUUU 157 ACUGUAGAAGGGCAUGAAGUU 158 D-1079 UUCAUGCCCUUCUACAGUGUU 159 CACUGUAGAAGGGCAUGAAUU 160 D-1080 AUGGCUUCCAGAUAUGCCUUU 161 AGGCAUAUCUGGAAGCCAUUU 162 D-1081 AUGCCCUUCUACAGUGGCCUU 163 GGCCACUGUAGAAGGGCAUUU 164 D-1082 GCUUCAUGCCCUUCUACAUUU 165 AUGUAGAAGGGCAUGAAGCUU 166 D-1083 GGAAAGACUGUUCCAAAAAUU 333 UUUUUGGAACAGUCUUUCCUU 334 D-1084 GGUAUGUUCCUGCUUCAUGUU 335 CAUGAAGCAGGAACAUACCUU 336 D-1085 GUAUGUUCCUGCUUCAUGCUU 337 GCAUGAAGCAGGAACAUACUU 338 D-1086 UGUUCCUGCUUCAUGCCCUUU 339 AGGGCAUGAAGCAGGAACAUU 340 D-1087 GCUUCAUGCCCUUCUACAGUU 341 CUGUAGAAGGGCAUGAAGCUU 342 D-1088 CUUCAUGCCCUUCUACAGUUU 343 ACUGUAGAAGGGCAUGAAGUU 344 D-1089 GCGGCUUCCUGGGCUUCUAUU 345 UAGAAGCCCAGGAAGCCGCUU 346 D-1090 GUUCCUGCUUCAUGCCCUUUU 347 AAGGGCAUGAAGCAGGAACUU 348 D-1091 AUGGCUUCCAGAUAUGCCUUU 349 AGGCAUAUCUGGAAGCCAUUU 350 D-1092 CCUGCUUCAUGCCCUUCUAUU 351 UAGAAGGGCAUGAAGCAGGUU 352 D-1093 UUCAUGCCCUUCUACAGUUUU 353 AACUGUAGAAGGGCAUGAAUU 354 D-1094 UUCAUGCCCUUCUACAGUGUU 355 CACUGUAGAAGGGCAUGAAUU 356 D-1095 GCUUCAUGCCCUUCUACAUUU 357 AUGUAGAAGGGCAUGAAGCUU 358 D-1096 GGUCCAGCCUGAACUUCUUUU 359 AAGAAGUUCAGGCUGGACCUU 360 D-1097 GCGGCUUCCUGGGCUUCUAUU 361 UAGAAGCCCAGGAAGCCGCUU 362 D-1098 GCGGCUUCCUGGGCUUCUAUU 363 UAGAAGCCCAGGAAGCCGCUU 364 D-1099 GUUCCUGCUUCAUGCCCUUUU 365 AAGGGCAUGAAGCAGGAACUU 366 D-1100 GUUCCUGCUUCAUGCCCUUUU 367 AAGGGCAUGAAGCAGGAACUU 368 D-1101 CCUGCUUCAUGCCCUUCUAUU 369 UAGAAGGGCAUGAAGCAGGUU 370 D-1102 CCUGCUUCAUGCCCUUCUAUU 371 UAGAAGGGCAUGAAGCAGGUU 372 D-1103 AUGCCCUUCUACAGUGGCCUU 373 GGCCACUGUAGAAGGGCAUUU 374 D-1104 AUGGCUUCCAGAUAUGCCUUU 375 AGGCAUAUCUGGAAGCCAUUU 376 D-1105 GUGACAACGUACCCUUCAUUU 377 AUGAAGGGUACGUUGUCACUU 378 D-1106 GUAUGUUCCUGCUUCAUGCUU 379 GCAUGAAGCAGGAACAUACUU 380 D-1107 GUAUGUUCCUGCUUCAUGCUU 381 GCAUGAAGCAGGAACAUACUU 382 D-1108 GUAUGUUCCUGCUUCAUGCUU 383 GCAUGAAGCAGGAACAUACUU 384 D-1109 GUAUGUUCCUGCUUCAUGCCU 385 AGGCAUGAAGCAGGAACAUACUU 386 D-1110 UGGUAUGUUCCUGCUUCAUGU 387 GCAUGAAGCAGGAACAUACCAUU 388 D-1111 GUAUGUUCCUGCUUCAUGU 389 GCAUGAAGCAGGAACAUACUU 390 D-1112 GUAUGUUCCUGCUUCAUGC{INVAB} 391 GCAUGAAGCAGGAACAUACUU 392 D-1113 GCUUCAUGCCCUUCUACAUUU 393 AUGUAGAAGGGCAUGAAGCUU 394 D-1114 GCUUCAUGCCCUUCUACAUUU 395 AUGUAGAAGGGCAUGAAGCUU 396 D-1115 GCUUCAUGCCCUUCUACAUUU 397 AUGUAGAAGGGCAUGAAGCUU 398 D-1116 GCUUCAUGCCCUUCUACAUUU 399 AUGUAGAAGGGCAUGAAGCUU 400 D-1117 GCUUCAUGCCCUUCUACAUUU 401 AUGUAGAAGGGCAUGAAGCUU 402 D-1118 GCUUCAUGCCCUUCUACAUUU 403 AUGUAGAAGGGCAUGAAGCUU 404 D-1119 GCUUCAUGCCCUUCUACAUUU 405 AUGUAGAAGGGCAUGAAGCUU 406 D-1120 GCUUCAUGCCCUUCUACAUUU 407 AUGUAGAAGGGCAUGAAGCUU 408 D-1121 GCUUCAUGCCCUUCUACAUUU 409 AUGUAGAAGGGCAUGAAGCUU 410 D-1122 GCUUCAUGCCCUUCUACAUUU 411 AUGUAGAAGGGCAUGAAGCUU 412 D-1123 GCUUCAUGCCCUUCUACAUUU 413 AUGUAGAAGGGCAUGAAGCUU 414 D-1124 GCUUCAUGCCCUUCUACAUUU 415 AUGUAGAAGGGCAUGAAGCUU 416 D-1125 GCUUCAUGCCCUUCUACAUUU 417 AUGUAGAAGGGCAUGAAGCUU 418 D-1126 GCUUCAUGCCCUUCUACAUUU 419 AUGUAGAAGGGCAUGAAGCUU 420 D-1127 GCUUCAUGCCCUUCUACAUUU 421 AUGUAGAAGGGCAUGAAGCUU 422 D-1128 GCUUCAUGCCCUUCUACAUUU 423 AUGUAGAAGGGCAUGAAGCUU 424 D-1129 GCUUCAUG[DC]CCUUCUACAUUU 425 AUGUAGAAGGGCAUGAAGCUU 426 D-1130 GCUUCAUGCC[DC]UUCUACAUUU 427 AUGUAGAAGGGCAUGAAGCUU 428 D-1131 GCUUCAUGC[DC]CUUCUACAUUU 429 AUGUAGAAGGGCAUGAAGCUU 430 D-1132 GCUUCAUGCCCUUCUACAUUU 431 AUGUAGAAGGGCAUGAAGCUU 432 D-1133 GCUUCAUGCCCUUCUACAUUU 433 AUGUAGAAGGGCAUGAAGCUU 434 D-1134 GCUUCAUGCCCUUCUACAUUU 435 AUGUAGAAGGGCAUGAAGCUU 436 D-1135 GCUUCAUGCCCUUCUACAUUU 437 AUGUAGAAGGGCAUGAAGCUU 438 D-1136 GCUUCAUGCCCUUCUACAUUU 439 AUGUAGAAGGGCAUGAAGCUU 440 D-1137 GCUUCAUGCCCUUCUACAGUU 441 AACUGUAGAAGGGCAUGAAGCUU 442 D-1138 CUGCUUCAUGCCCUUCUACAU 443 AUGUAGAAGGGCAUGAAGCAGUU 444 D-1139 GCUUCAUGCCCUUCUACAU 445 AUGUAGAAGGGCAUGAAGCUU 446 D-1140 GCUUCAUGCCCUUCUACAU{INVAB} 447 AUGUAGAAGGGCAUGAAGCUU 448 D-1141 GCUUCAUGCCCUUCUACAUUU{INVAB} 449 AUGUAGAAGGGCAUGAAGCUU 450 D-1142 GCUUCAUGCCCUUCUACAUUU 451 AUGUAGAAGGGCAUGAAGCUU 452 D-1143 GCUUCAUGCCCUUCUACAUUU 453 AUGUAGAAGGGCAUGAAGCUU 454 D-1144 GCUUCAUGCCCUUCUACAUUU 455 AUGUAGAAGGGCAUGAAGCUU 456 D-1145 GCUUCAUGCCCUUCUACAUUU 457 AUGUAGAAGGGCAUGAAGCUU 458 D-1146 GCUUCAUGCCCUUCUACAUUU 459 AUGUAGAAGGGCAUGAAGCUU 460 D-1147 GCUUCAUGCCCUUCUACAUUU 461 AUGUAGAAGGGCAUGAAGCUU 462 D-1148 GCUUCAUGCCCUUCUACAUUU 463 AUGUAGAAGGGCAUGAAGCUU 464 D-1149 GCUUCAUGCCCUUCUACAUUU 465 AUGUAGAAGGGCAUGAAGCUU 466 D-1150 GCUUCAUGCCCUUCUACAUUU 467 AUGUAGAAGGGCAUGAAGCUU 468 D-1151 GUAUGUUCCUGCUUCAUGCUU{INVAB} 469 GCAUGAAGCAGGAACAUACUU 470 D-1152 GGUAUGUUCCUGCUUCAUUUU 471 AAUGAAGCAGGAACAUACCUU 472 D-1153 GUAUGUUCCUGCUUCAUGUUU 473 ACAUGAAGCAGGAACAUACUU 474 D-1154 CGGCCAAUGUCCACCAGCUUU 475 AGCUGGUGGACAUUGGCCGUU 476 D-1155 UGGAGCAGACUCUGCAGGUUU 477 ACCUGCAGAGUCUGCUCCAUU 478 D-1156 ACGUACCCUUCAUUGAUGUUU 479 ACAUCAAUGAAGGGUACGUUU 480 D-1157 CCAGAGACUGGUGACAUGUUU 481 ACAUGUCACCAGUCUCUGGUU 482 D-1158 [INVAB]GCUUCAUGCCUUUCUACAUUU 483 AUGUAGAAAGGCAUGAAGCUU 484 D-1159 [INVAB]GCUUCAUGCCUUUCUACAUUU 485 AAAUGUAGAAAGGCAUGAAGCUU 486 D-1160 [INVAB]GCUUCAUGCCUUUCUACAUUU 487 AAAUGUAGAAAGGCAUGAAGCUU 488 D-1161 UGCUUCAUGCCUUUCUACAUU 489 UGUAGAAAGGCAUGAAGCAUU 490 D-1162 UAUGUUCCUGCUUCAUGCUUU 491 AGCAUGAAGCAGGAACAUAUU 492 D-1163 UUCCUGCUUCAUGCCUUUUUU 493 AAAAGGCAUGAAGCAGGAAUU 494 D-1164 UCAUGCCUUUCUACAGUGUUU 495 ACACUGUAGAAAGGCAUGAUU 496 D-1165 CAUGCCUUUCUACAGUGGUUU 497 ACCACUGUAGAAAGGCAUGUU 498 D-1166 AUGCCUUUCUACAGUGGCUUU 499 AGCCACUGUAGAAAGGCAUUU 500 D-1167 GGUAUGUUCCUGCUUCAUAUU 501 UAUGAAGCAGGAACAUACCUU 502 D-1168 GUAUGUUCCUGCUUCAUGAUU 503 UCAUGAAGCAGGAACAUACUU 504 D-1169 UAUGUUCCUGCUUCAUGCAUU 505 UGCAUGAAGCAGGAACAUAUU 506 D-1170 UUCCUGCUUCAUGCCUUUAUU 507 UAAAGGCAUGAAGCAGGAAUU 508 D-1171 CUGCUUCAUGCCUUUCUAAUU 509 UUAGAAAGGCAUGAAGCAGUU 510 D-1172 GCUUCAUGCCUUUCUACAAUU 511 UUGUAGAAAGGCAUGAAGCUU 512 D-1173 UUCAUGCCUUUCUACAGUAUU 513 UACUGUAGAAAGGCAUGAAUU 514 D-1174 UCAUGCCUUUCUACAGUGAUU 515 UCACUGUAGAAAGGCAUGAUU 516 D-1175 CAUGCCUUUCUACAGUGGAUU 517 UCCACUGUAGAAAGGCAUGUU 518 D-1176 AUGCCUUUCUACAGUGGCAUU 519 UGCCACUGUAGAAAGGCAUUU 520 D-1177 ACGUACCCUUCAUUGAUGAUU 521 UCAUCAAUGAAGGGUACGUUU 522 D-1178 CCAGAGACUGGUGACAUGAUU 523 UCAUGUCACCAGUCUCUGGUU 524 D-1179 AUGGCUUCCAGAUAUGCCAUU 525 UGGCAUAUCUGGAAGCCAUUU 526 D-1180 GUUCCUGCUUCAUGCCUUUUU 527 AAAGGCAUGAAGCAGGAACUU 528 D-1181 CCUGCUUCAUGCCUUUCUAUU 529 UAGAAAGGCAUGAAGCAGGUU 530 D-1182 GCUUCAUGCCUUUCUACAUUU 531 AUGUAGAAAGGCAUGAAGCUU 532 D-1183 CUUCAUGCCUUUCUACAGUUU 533 ACUGUAGAAAGGCAUGAAGUU 534 D-1184 UUCAUGCCUUUCUACAGUUUU 535 AACUGUAGAAAGGCAUGAAUU 536 D-1185 GCUUCAUCCCUUUCUACAUUU 537 AUGUAGAAAGGGAUGAAGCUU 538 D-1186 [INVAB]GCUUCAUGCCCUUCUACAUUU 539 AUGUAGAAGGGCAUGAAGCUU 540 D-1187 [INVAB]GCUUCAUGCCCUUCUACAUUU 541 AAAUGUAGAAGGGCAUGAAGCUU 542 D-1188 [INVAB]GCUUCAUGCCCUUCUACAUUU 543 AAAUGUAGAAGGGCAUGAAGCUU 544 D-1189 [INVAB]GCGGCUUCCUGGGCUUCUAUU 545 UAGAAGCCCAGGAAGCCGCUU 546 D-1190 [INVAB]CUGCGGCUUCCUGGGCUUCUA 547 UAGAAGCCCAGGAAGCCGCAGUU 548 D-1191 CUGCGGCUUCCUGGGCUUCU{INVAB} 549 UAGAAGCCCAGGAAGCCGCAGUU 550 D-1192 [INVAB]CUGCGGCUUCCUGGGCUUCUA 551 UAGAAGCCCAGGAAGCCGCAGUU 552 D-1193 [INVAB]GCGGCUUCCUGGGCUUCUAUU 553 UAGAAGCCCAGGAAGCCGCUU 554 D-1194 CUGCGGCUUCCUGGGCUUCU{INVAB} 555 UAGAAGCCCAGGAAGCCGCAGUU 556 D-1195 [INVAB]CUGCGGCUUCCUGGGCUUCUA 557 UAGAAGCCCAGGAAGCCGCAGUU 558 D-1196 [INVAB]GCGGCUUCCUGGGCUUCUAUU 559 UAGAAGCCCAGGAAGCCGCUU 560 D-1197 CUGCGGCUUCCUGGGCUUCU{INVAB} 561 UAGAAGCCCAGGAAGCCGCAGUU 562 D-1198 [INVAB]CUGCGGCUUCCUGGGCUUCUA 563 UAGAAGCCCAGGAAGCCGCAGUU 564 D-1199 [INVAB]GCGGCUUCCUGGGCUUCUAUU 565 UAGAAGCCCAGGAAGCCGCUU 566 D-1200 [INVAB]CUGCGGCUUCCUGGGCUUCUA 567 UAGAAGCCCAGGAAGCCGCAGUU 568 D-1201 [INVAB]CUGCGGCUUCCUGGGCUUCUA 569 UAGAAGCCCAGGAAGCCGCAGUU 570 D-1202 [INVAB]CUGCGGCUUCCUGGGCUUCUA 571 UAGAAGCCCAGGAAGCCGCAGUU 572 D-1203 [INVAB]AUGGCUUCCAGAUAUGCCUUU 573 AGGCAUAUCUGGAAGCCAUUU 574 D-1204 [INVAB]ACAUGGCUUCCAGAUAUGCCU 575 AGGCAUAUCUGGAAGCCAUGUUU 576 D-1205 ACAUGGCUUCCAGAUAUGCC{INVAB} 577 AGGCAUAUCUGGAAGCCAUGUUU 578 D-1206 [INVAB]ACAUGGCUUCCAGAUAUGCCU 579 AGGCAUAUCUGGAAGCCAUGUUU 580 D-1207 [INVAB]AUGGCUUCCAGAUAUGCCUUU 581 AGGCAUAUCUGGAAGCCAUUU 582 D-1208 ACAUGGCUUCCAGAUAUGCC{INVAB} 583 AGGCAUAUCUGGAAGCCAUGUUU 584 D-1209 [INVAB]ACAUGGCUUCCAGAUAUGCCU 585 AGGCAUAUCUGGAAGCCAUGUUU 586 D-1210 [INVAB]AUGGCUUCCAGAUAUGCCUUU 587 AGGCAUAUCUGGAAGCCAUUU 588 D-1211 ACAUGGCUUCCAGAUAUGCC{INVAB} 589 AGGCAUAUCUGGAAGCCAUGUUU 590 D-1212 [INVAB]ACAUGGCUUCCAGAUAUGCCU 591 AGGCAUAUCUGGAAGCCAUGUUU 592 D-1213 [INVAB]AUGGCUUCCAGAUAUGCCUUU 593 AGGCAUAUCUGGAAGCCAUUU 594 D-1214 ACAUGGCUUCCAGAUAUGCC{INVAB} 595 AGGCAUAUCUGGAAGCCAUGUUU 596 D-1215 [INVAB]ACAUGGCUUCCAGAUAUGCCU 597 AGGCAUAUCUGGAAGCCAUGUUU 598 D-1216 [INVAB]ACAUGGCUUCCAGAUAUGCCU 599 AGGCAUAUCUGGAAGCCAUGUUU 600 D-1217 [INVAB]ACAUGGCUUCCAGAUAUGCCU 601 AGGCAUAUCUGGAAGCCAUGUUU 602 D-1218 [INVAB]ACAUGGCUUCCAGAUAUGCCU 603 AGGCAUAUCUGGAAGCCAUGUUU 604 D-1219 [INVAB]ACGUACCCUUCAUUGAUGUUU 605 ACAUCAAUGAAGGGUACGUUU 606 D-1220 [INVAB]CAACGUACCCUUCAUUGAUGU 607 ACAUCAAUGAAGGGUACGUUGUU 608 D-1221 [INVAB]CAACGUACCCUUCAUUGAUGU 609 ACAUCAAUGAAGGGUACGUUGUU 610 D-1222 [INVAB]ACGUACCCUUCAUUGAUGUUU 611 ACAUCAAUGAAGGGUACGUUU 612 D-1223 CAACGUACCCUUCAUUGAUG{INVAB} 613 ACAUCAAUGAAGGGUACGUUGUU 614 D-1224 [INVAB]ACGUACCCUUCAUUGAUGUUU 615 ACAUCAAUGAAGGGUACGUUU 616 D-1225 CAACGUACCCUUCAUUGAUG{INVAB} 617 ACAUCAAUGAAGGGUACGUUGUU 618 D-1226 [INVAB]CAACGUACCCUUCAUUGAUGU 619 ACAUCAAUGAAGGGUACGUUGUU 620 D-1227 [INVAB]ACGUACCCUUCAUUGAUGUUU 621 ACAUCAAUGAAGGGUACGUUU 622 D-1228 CAACGUACCCUUCAUUGAUG{INVAB} 623 ACAUCAAUGAAGGGUACGUUGUU 624 D-1229 [INVAB]CAACGUACCCUUCAUUGAUGU 625 ACAUCAAUGAAGGGUACGUUGUU 626 D-1230 [INVAB]CAACGUACCCUUCAUUGAUGU 627 ACAUCAAUGAAGGGUACGUUGUU 628 D-1231 [INVAB]CAACGUACCCUUCAUUGAUGU 629 ACAUCAAUGAAGGGUACGUUGUU 630 D-1232 [INVAB]CAACGUACCCUUCAUUGAUGU 631 ACAUCAAUGAAGGGUACGUUGUU 632 D-1233 CUGCUUCAUGCCUUUCUACAU 633 AUGUAGAAAGGCAUGAAGCAGUU 634 D-1234 CUGCUUCAUGCCUUUCUACAU 635 AUGUAGAAAGGCAUGAAGCAGUU 636 D-1235 [INVAB]GCUUCAUGCCUUUCUACAUUU 637 AUGUAGAAAGGCAUGAAGCUU 638 D-1236 [INVAB]GCUUCAUGCCUUUCUACAUUU 639 AUGUAGAAAGGCAUGAAGCUU 640 D-1237 CUGCUUCAUGCCUUUCUACAU 641 AUGUAGAAAGGCAUGAAGCAGUU 642 D-1238 CUGCUUCAUGCCUUUCUACAU 643 AUGUAGAAAGGCAUGAAGCAGUU 644 D-1239 [INVAB]CUGCUUCAUGCCUUUCUACAU 645 AUGUAGAAAGGCAUGAAGCAGUU 646 D-1240 CUGCUUCAUGCCUUUCUACA{INVAB} 647 AUGUAGAAAGGCAUGAAGCAGUU 648 D-1241 [INVAB]CUGCUUCAUGCCUUUCUACAU 649 AUGUAGAAAGGCAUGAAGCAGUU 650 D-1242 [INVAB]CUGCUUCAUGCUUUCUACAU 651 AUGUAGAAAGGCAUGAAGCAGUU 652 D-1243 CUGCUUCAUGCCUUUCUACAU 653 AUGUAGAAAGGCAUGAAGCAGUU 654 D-1244 [INVAB]CUGCUUCAUGCCUUUCUACAU 655 AUGUAGAAAGGCAUGAAGCAGUU 656 D-1245 CUGCUUCAUGCCUUUCUACAU 657 AUGUAGAAAGGCAUGAAGCAGUU 658 D-1246 CUGCUUCAUGCCUUUCUACA{INVAB} 659 AUGUAGAAAGGCAUGAAGCAGUU 660 D-1247 [INVAB]CUGCUUCAUGCCUUUCUACAU 661 AUGUAGAAAGGCAUGAAGCAGUU 662 D-1248 CUGCUUCAUGCCUUUCUACAU 663 AUGUAGAAAGGCAUGAAGCAGUU 664 D-1249 CUGCUUCAUGCCUUUCUACA{INVAB} 665 AUGUAGAAAGGCAUGAAGCAGUU 666 D-1250 [INVAB]CUGCUUCAUGCCUUUCUACAU 667 AUGUAGAAAGGCAUGAAGCAGUU 668 D-1251 CUGCUUCAUGCCUUUCUACA{INVAB} 669 AUGUAGAAAGGCAUGAAGCAGUU 670 D-1252 [INVAB]CUGCUUCAUGCCUUUCUACAU 671 AUGUAGAAAGGCAUGAAGCAGUU 672 D-1253 [INVAB]GCUUCAUGCCUUUCUACAUUU 673 AUGUAGAAAGGCAUGAAGCUU 674 D-1254 [INVAB]GCUUCAUGCCUUUCUACAUUU 675 AUGUAGAAAGGCAUGAAGCUU 676 D-1255 [INVAB]GCUUCAUGCCUUUCUACAUUU 677 AUGUAGAAAGGCAUGAAGCUU 678 D-1256 [INVAB]GCUUCAUGCCUUUCUACAUUU 679 AUGUAGAAAGGCAUGAAGCUU 680 D-1257 [INVAB]GCUUCAUGCCUUUCUACAUUU 681 AUGUAGAAAGGCAUGAAGCUU 682 D-1258 CUGCUUCAUGCCCUUCUACAU 683 AUGUAGAAGGGCAUGAAGCAGUU 684 D-1259 [INVAB]GCUUCAUGCCCUUCUACAUUU 685 AUGUAGAAGGGCAUGAAGCUU 686 D-1260 [INVAB]GCUUCAUGCCCUUCUACAUUU 687 AUGUAGAAGGGCAUGAAGCUU 688 D-1261 [INVAB]GCUUCAUGCCCUUCUACAUUU 689 AUGUAGAAGGGCAUGAAGCUU 690 D-1262 CUGCUUCAUGCCCUUCUACA{INVAB} 691 AUGUAGAAGGGCAUGAAGCAGUU 692 D-1263 [INVAB]CUGCUUCAUGCCCUUCUACAU 693 AUGUAGAAGGGCAUGAAGCAGUU 694 D-1264 [INVAB]GCUUCAUGCCCUUCUACAUUU 695 AUGUAGAAGGGCAUGAAGCUU 696 D-1265 AUGUUCCUGCUUCAUGCCUUU 697 AGGCAUGAAGCAGGAACAUUU 698 D-1266 UGUUCCUGCUUCAUGCCUUUU 699 AAGGCAUGAAGCAGGAACAUU 700 D-1267 UGCCUUUCUACAGUGGCCUUU 701 AGGCCACUGUAGAAAGGCAUU 702 D-1268 CGUACUUCGUCCUUGUAUGUU 703 CAUACAAGGACGAAGUACGUU 704 D-1269 [INVAB]GCUUCAUGCCUUCUACAUUU 705 AUGUAGAAGGGCAUGAAGCUU 706 D-1270 [INVAB]GCUUCAUGCCCUUCUACAUUU 707 AUGUAGAAGGGCAUGAAGCUU 708 D-1271 [INVAB]GCUUCAUGCCCUUCUACAUUU 709 AUGUAGAAGGGCAUGAAGCUU 710 D-1272 [INVAB]GCUUCAUGCCCUUCUACAUUU 711 AUGUAGAAGGGCAUGAAGCUU 712 D-1273 [INVAB]GCUUCAUGCCCUUCUACAUUU 713 AUGUAGAAGGGCAUGAAGCUU 714 D-1274 [INVAB]GCUUCAUGCCCUUCUACAUUU 715 AUGUAGAAGGGCAUGAAGCUU 716 D-1275 [INVAB]CCUGCUUCAUGCCUUUCUAUU 717 UAGAAAGGCAUGAAGCAGGUU 718 D-1276 [INVAB]CCUGCUUCAUGCCUUUCUAUU 719 UAGAAAGGCAUGAAGCAGGUU 720 D-1277 UUCCUGCUUCAUGCCUUUCU{INVAB} 721 UAGAAAGGCAUGAAGCAGGAAUU 722 D-1278 [INVAB]CCUGCUUCAUGCCUUUCUAUU 723 UAGAAAGGCAUGAAGCAGGUU 724 D-1279 UUCCUGCUUCAUGCCUUUCU{INVAB} 725 UAGAAAGGCAUGAAGCAGGAAUU 726 D-1280 [INVAB]AUGCCUUUCUACAGUGGCUUU 727 AGCCACUGUAGAAAGGCAUUU 728 D-1281 [INVAB]AUGCCUUUCUACAGUGGCUUU 729 AGCCACUGUAGAAAGGCAUUU 730 D-1282 [INVAB]AUGCCUUUCUACAGUGGCUUU 731 AGCCACUGUAGAAAGGCAUUU 732 D-1283 UCAUGCCUUUCUACAGUGGC{INVAB} 733 AGCCACUGUAGAAAGGCAUGAUU 734 D-1284 [INVAB]AUGCCUUUCUACAGUGGCUUU 735 AGCCACUGUAGAAAGGCAUUU 736 D-1285 [INVAB]UCAUGCCUUUCUACAGUGGCU 737 AGCCACUGUAGAAAGGCAUGAUU 738 D-1286 [INVAB]UGCUUCAUGCCUUUCUACAGU 739 ACUGUAGAAAGGCAUGAAGCAUU 740 D-1287 [INVAB]CUUCAUGCCUUUCUACAGUUU 741 ACUGUAGAAAGGCAUGAAGUU 742 D-1289 UGCUUCAUGCCUUUCUACAG{INVAB} 743 ACUGUAGAAAGGCAUGAAGCAUU 744 D-1290 [INVAB]CUUCAUGCCUUUCUACAGUUU 745 ACUGUAGAAAGGCAUGAAGUU 746 D-1291 UGCUUCAUGCCUUUCUACAG{INVAB} 747 ACUGUAGAAAGGCAUGAAGCAUU 748 D-1292 [INVAB]UGCUUCAUGCCUUUCUACAGU 749 ACUGUAGAAAGGCAUGAAGCAUU 750 D-1293 [INVAB]UGCUUCAUGCCUUUCUACAGU 751 ACUGUAGAAAGGCAUGAAGCAUU 752 D-1294 [INVAB]UGCUUCAUGCCUUUCUACAGU 753 ACUGUAGAAAGGCAUGAAGCAUU 754 D-1295 [INVAB]UGCUUCAUGCCUUUCUACAGU 755 ACUGUAGAAAGGCAUGAAGCAUU 756 D-1296 [INVAB]GUUCCUGCUUCAUGCCUUUUU 757 AAAGGCAUGAAGCAGGAACUU 758 D-1297 [INVAB]CCUGCUUCAUGCCUUUCUAUU 759 UAGAAAGGCAUGAAGCAGGUU 760 D-1298 [INVAB]CUUCAUGCCUUUCUACAGUUU 761 ACUGUAGAAAGGCAUGAAGUU 762 D-1299 [INVAB]UUCCUGCUUCAUGCCUUUCUA 763 UAGAAAGGCAUGAAGCAGGAAUU 764 D-1300 UUCCUGCUUCAUGCCUUUCU{INVAB} 765 UAGAAAGGCAUGAAGCAGGAAUU 766 D-1301 [INVAB]UUCCUGCUUCAUGCCUUUCUA 767 UAGAAAGGCAUGAAGCAGGAAUU 768 D-1302 UUCCUGCUUCAUGCCUUUCU{INVAB} 769 UAGAAAGGCAUGAAGCAGGAAUU 770 D-1303 [INVAB]UUCCUGCUUCAUGCCUUUCUA 771 UAGAAAGGCAUGAAGCAGGAAUU 772 D-1304 [INVAB]UUCCUGCUUCAUGCCUUUCUA 773 UAGAAAGGCAUGAAGCAGGAAUU 774 D-1305 [INVAB]UUCCUGCUUCAUGCCUUUCUA 775 UAGAAAGGCAUGAAGCAGGAAUU 776 D-1306 [INVAB]UUCCUGCUUCAUGCCUUUCUA 777 UAGAAAGGCAUGAAGCAGGAAUU 778 D-1307 [INVAB]UUCCUGCUUCAUGCCUUUCUA 779 UAGAAAGGCAUGAAGCAGGAAUU 780 D-1308 [INVAB]UUCCUGCUUCAUGCCUUUCUA 781 UAGAAAGGCAUGAAGCAGGAAUU 782 D-1309 [INVAB]UCAUGCCUUUCUACAGUGGCU 783 AGCCACUGUAGAAAGGCAUGAUU 784 D-1310 UCAUGCCUUUCUACAGUGGC{INVAB} 785 AGCCACUGUAGAAAGGCAUGAUU 786 D-1311 [INVAB]UCAUGCCUUUCUACAGUGGCU 787 AGCCACUGUAGAAAGGCAUGAUU 788 D-1312 UCAUGCCUUUCUACAGUGGC{INVAB} 789 AGCCACUGUAGAAAGGCAUGAUU 790 D-1313 [INVAB]UCAUGCCUUUCUACAGUGGCU 791 AGCCACUGUAGAAAGGCAUGAUU 792 D-1314 [INVAB]UCAUGCCUUUCUACAGUGGCU 793 AGCCACUGUAGAAAGGCAUGAUU 794 D-1315 UCAUGCCUUUCUACAGUGGC{INVAB} 795 AGCCACUGUAGAAAGGCAUGAUU 796 D-1316 [INVAB]UCAUGCCUUUCUACAGUGGCU 797 AGCCACUGUAGAAAGGCAUGAUU 798 D-1317 [INVAB]UCAUGCCUUUCUACAGUGGCU 799 AGCCACUGUAGAAAGGCAUGAUU 800 D-1318 [INVAB]UCAUGCCUUUCUACAGUGGCU 801 AGCCACUGUAGAAAGGCAUGAUU 802 D-1319 [INVAB]UGCUUCAUGCCUUUCUACAGU 803 ACUGUAGAAAGGCAUGAAGCAUU 804 D-1320 UGCUUCAUGCCUUUCUACAG{INVAB} 805 ACUGUAGAAAGGCAUGAAGCAUU 806 D-1321 UGCUUCAUGCCUUUCUACAG{INVAB} 807 ACUGUAGAAAGGCAUGAAGCAUU 808 D-1322 [INVAB]UGCUUCAUGCCUUUCUACAGU 809 ACUGUAGAAAGGCAUGAAGCAUU 810 D-1323 [INVAB]CUUCAUGCCUUUCUACAGUUU 811 ACUGUAGAAAGGCAUGAAGUU 812 D-1324 [INVAB]UGCUUCAUGCCUUUCUACAGU 813 ACUGUAGAAAGGCAUGAAGCAUU 814 D-1325 [INVAB]GCUUCAUGCCUUUCUACAUUU 815 AUGUAGAAAGGCAUGAAGCUU 816 D-1326 [INVAB]CAUGCCUUUCUACAGUGGUUU 817 ACCACUGUAGAAAGGCAUGUU 818 D-1327 [INVAB]CUGCUUCAUGCCUUUCUAAUU 819 UUAGAAAGGCAUGAAGCAGUU 820 D-1328 [INVAB]GCUUCAUGCCUUUCUACAAUU 821 UUGUAGAAAGGCAUGAAGCUU 822 D-1329 [INVAB]UUCAUGCCUUUCUACAGUAUU 823 UACUGUAGAAAGGCAUGAAUU 824 D-1330 [INVAB]GUUCCUGCUUCAUGCCUUUUU 825 AAAGGCAUGAAGCAGGAACUU 826 D-1331 AUGUUCCUGCUUCAUGCCUU{INVAB} 827 AAAGGCAUGAAGCAGGAACAUUU 828 D-1332 [INVAB]AUGUUCCUGCUUCAUGCCUUU 829 AAAGGCAUGAAGCAGGAACAUUU 830 D-1333 [INVAB]GUUCCUGCUUCAUGCCUUUUU 831 AAAGGCAUGAAGCAGGAACUU 832 D-1334 AUGUUCCUGCUUCAUGCCUU{INVAB} 833 AAAGGCAUGAAGCAGGAACAUUU 834 D-1335 [INVAB]AUGUUCCUGCUUCAUGCCUUU 835 AAAGGCAUGAAGCAGGAACAUUU 836 D-1336 [INVAB]GUUCCUGCUUCAUGCCUUUUU 837 AAAGGCAUGAAGCAGGAACUU 838 D-1337 AUGUUCCUGCUUCAUGCCUU{INVAB} 839 AAAGGCAUGAAGCAGGAACAUUU 840 D-1338 [INVAB]AUGUUCCUGCUUCAUGCCUUU 841 AAAGGCAUGAAGCAGGAACAUUU 842 D-1339 [INVAB]AUGUUCCUGCUUCAUGCCUUU 843 AAAGGCAUGAAGCAGGAACAUUU 844 D-1340 [INVAB]AUGUUCCUGCUUCAUGCCUUU 845 AAAGGCAUGAAGCAGGAACAUUU 846 D-1341 [INVAB]GCUUCAUGCCUUUCUACAGUA 847 UACUGUAGAAAGGCAUGAAGCUU 848 D-1342 [INVAB]GCUUCAUGCCUUUCUACAGUA 849 UACUGUAGAAAGGCAUGAAGCUU 850 D-1343 [INVAB]UUCAUGCCUUUCUACAGUAUU 851 UACUGUAGAAAGGCAUGAAUU 852 D-1344 [INVAB]GCUUCAUGCCUUUCUACAGUA 853 UACUGUAGAAAGGCAUGAAGCUU 854 D-1345 GCUUCAUGCCUUUCUACAGU{INVAB} 855 UACUGUAGAAAGGCAUGAAGCUU 856 D-1346 [INVAB]GCGGCUUCCUGGGCUUCUAUU 857 UAGAAGCCCAGGAAGCCGCUU 858 D-1347 [INVAB]AUGGCUUCCAGAUAUGCCUUU 859 AGGCAUAUCUGGAAGCCAUUU 860 D-1348 [INVAB]ACAUGGCUUCCAGAUAUGCCU 861 AGGCAUAUCUGGAAGCCAUGUUU 862 D-1349 [INVAB]ACAUGGCUUCCAGAUAUGCCU 863 AGGCAUAUCUGGAAGCCAUGUUU 864 D-1350 [INVAB]CAACGUACCCUUCAUUGAUGU 865 ACAUCAAUGAAGGGUACGUUGUU 866 D-1351 [INVAB]CAACGUACCCUUCAUUGAUGU 867 ACAUCAAUGAAGGGUACGUUGUU 868 D-1352 [INVAB]ACGUACCCUUCAUUGAUGUUU 869 ACAUCAAUGAAGGGUACGUUU 870 D-1353 CUGCUUCAUGCCUUUCUACA{INVAB} 871 AUGUAGAAAGGCAUGAAGCAGUU 872 D-1354 [INVAB]CUGCUUCAUGCCUUUCUACAU 873 AUGUAGAAAGGCAUGAAGCAGUU 874 D-1355 [INVAB]GCUUCAUGCCUUUCUACAUUU 875 AUGUAGAAAGGCAUGAAGCUU 876 D-1356 CUGCUUCAUGCCUUUCUACA{INVAB} 877 AUGUAGAAAGGCAUGAAGCAGUU 878 D-1357 [INVAB]CUGCUUCAUGCCUUUCUACAU 879 AUGUAGAAAGGCAUGAAGCAGUU 880 D-1358 CUGCUUCAUGCCUUUCUACA{INVAB} 881 AUGUAGAAAGGCAUGAAGCAGUU 882 D-1359 [INVAB]CUGCUUCAUGCCUUUCUACA{INVAB} 883 AUGUAGAAAGGCAUGAAGCAGUU 884 D-1360 [INVAB]GCUUCAUGCCUUUCUACAUUU 885 AUGUAGAAAGGCAUGAAGCUU 886 D-1361 [INVAB]CUUCAUGCCUUUCUACAGUUU 887 ACUGUAGAAAGGCAUGAAGUU 888 D-1362 UGCUUCAUGCCUUUCUACAG{INVAB} 889 ACUGUAGAAAGGCAUGAAGCAUU 890 D-1363 [INVAB]CUGCUUCAUGCCUUUCUACAU 891 AUGUAGAAAGGCAUGAAGCAGUU 892 D-1364 CUGCUUCAUGCCUUUCUACA{INVAB} 893 AUGUAGAAAGGCAUGAAGCAGUU 894 D-1365 CUGCUUCAUGCCUUUCUACA{INVAB} 895 AUGUAGAAAGGCAUGAAGCAGUU 896 D-1366 [INVAB]CUUCAUCCCUUUCUACAGUUU 897 ACUGUAGAAAGGGAUGAAGUU 898 D-1367 [INVAB]GCUUCAUCCCUUUCUACAUUU 899 AUGUAGAAAGGGAUGAAGCUU 900 D-1368 UGCUUCAUCCCUUUCUACAG{INVAB} 901 ACUGUAGAAAGGGAUGAAGCAUU 902 D-1369 CUGCUUCAUCCCUUUCUACA{INVAB} 903 AUGUAGAAAGGGAUGAAGCAGUU 904 D-1370 [INVAB]ACAUUGCUCUUUCACCUGAUU 905 UCAGGUGAAAGAGCAAUGUUU 906 D-1371 CUGCUUCAUGCCUUUCUACA{INVAB} 907 AUGUAGAAAGGCAUGAAGCAGUU 908 D-1372 CUGCUUCAUGCCUUUCUACA{INVAB} 909 AUGUAGAAAGGCAUGAAGCAGUU 910 D-1373 CUGCUUCAUGCCUUUCUACA{INVAB} 911 AUGUAGAAAGGCAUGAAGCAGUU 912 D-1374 CUGCUUCAUGCCUUUCUACA{INVAB} 913 AUGUAGAAAGGCAUGAAGCAGUU 914 D-1375 CUGCUUCAUGCCUUUCUACA{INVAB} 915 AUGUAGAAAGGCAUGAAGCAGUU 916 D-1376 CUGCUUCAUGCCUUUCUACA{INVAB} 917 AUGUAGAAAGGCAUGAAGCAGUU 918 D-1377 CUGCUUCAUGCCUUUCUACA{INVAB} 919 AUGUAGAAAGGCAUGAAGCAGUU 920 D-1378 CUGCUUCAUGCCUUUCUACA{INVAB} 921 AUGUAGAAAGGCAUGAAGCAGUU 922 D-1379 CUGCUUCAUGCCUUUCUACA{INVAB} 923 AUGUAGAAAGGCAUGAAGCAGUU 924 D-1380 CUGCUUCAUGCCUUUCUACA{INVAB} 925 AUGUAGAAAGGCAUGAAGCAGUU 926 D-1381 CUGCUUCAUGCCUUUCUACA{INVAB} 927 AUGUAGAAAGGCAUGAAGCAGUU 928 D-1381 [INVAB]CUUCAUGCC[DT]UUCUACAGUUU 929 ACUGUAGAAAGGCAUGAAGUU 930 D-1382 [INVAB]CUUCAUGCCUUUCUACAGUUU 931 ACUGUAGAAAGGCAUGAAGUU 932 D-1383 [INVAB]CUUCAUGCCUUUCUACAGUUU 933 ACUGUAGAAAGGCAUGAAGUU 934 D-1384 [INVAB]CUUCAUGCCUUUCUACAGUUU 935 ACUGUAGAAAGGCAUGAAGUU 936 D-1385 [INVAB]CUUCAUGCCUUUCUACAGUUU 937 ACUGUAGAAAGGCAUGAAGUU 938 D-1386 [INVAB]CUUCAUGC[DC]UUUCUACAGUUU 939 ACUGUAGAAAGGCAUGAAGUU 940 D-1387 [INVAB]CUUCAUGCCU[DT]UCUACAGUUU 941 ACUGUAGAAAGGCAUGAAGUU 942 D-1388 [INVAB]GCUUCAUGGGAUUCUACAUUU 943 AUGUAGAAUCCCAUGAAGCUU 944 D-1389 [INVAB]CUUCAUGCGAAUCUACAGUUU 945 ACUGUAGAUUCGCAUGAAGUU 946 D-1390 CUGCUUCAUGCCUUUCUACA{INVAB} 947 UUGUAGAAAGGCAUGAAGCAGUU 948 D-1391 [INVAB]CUGCUUCAUGCCUUUCUACAA 949 UUGUAGAAAGGCAUGAAGCAGUU 950 D-1392 CUGCUUCAUGCCUUUCUACA{INVDA} 951 UUGUAGAAAGGCAUGAAGCAGUU 952 D-1393 CUGCUUCAUGGGAUUCUACA{INVAB} 953 AUGUAGAAUCCCAUGAAGCAGUU 954 D-1394 [INVAB]CUGCUUCAUGGGAUUCUACAU 955 AUGUAGAAUCCCAUGAAGCAGUU 956 D-1395 [INVAB]CUUCAUGCCUUUCUACAGUUU 957 ACUGUAGAAAGGCAUGAAGUU 958 D-1396 [INVAB]CUUCAUGCCUUUCUACAGUUU 959 ACUGUAGAAAGGCAUGAAGUU 960 D-1397 [INVAB]GCUUCAUGCCUUUCUACAUUU 961 AUGUAGAAAGGCAUGAAGCUU 962 D-1398 [INVAB]UGCUUCAUGCCUUUCUACAG{INVAB} 963 ACUGUAGAAAGGCAUGAAGCAUU 964 D-1399 [INVAB]CUUCAUGCCUUUCUACAGUUU 965 ACUGUAGAAAGGCAUGAAGUU 966 D-1400 UGCUUCAUGCCUUUCUACAG{INVAB} 967 ACUGUAGAAAGGCAUGAAGCAUU 968 D-1401 UGCUUCAUGCCUUUCUACAG{INVAB} 969 ACUGUAGAAAGGCAUGAAGCAUU 970 D-1402 UGCUUCAUGCCUUUCUACAG{INVAB} 971 ACUGUAGAAAGGCAUGAAGCAUU 972 D-1403 [INVAB]CUUCAUGCCUUUCUACAGUUU 973 ACUGUAGAAAGGCAUGAAGUU 974 D-1404 UGCUUCAUGCCUUUCUACAG{INVAB} 975 ACUGUAGAAAGGCAUGAAGCAUU 976 D-1405 AUGCCUUUCUACAGUGGCUU{INVAB} 977 AGCCACUGUAGAAAGGCAUGAUU 978 D-1406 UCAUGCCUUUCUACAGUGGC{INVAB} 979 AGCCACUGUAGAAAGGCAUGAUU 980 D-1407 [INVAB]AUGCCUUUCUACAGUGGCUUU 981 AGCCACUGUAGAAAGGCAUUU 982 D-1408 [INVAB]AUGCCUUUCUACAGUGGCUUU 983 AGCCACUGUAGAAAGGCAUUU 984 D-1409 [INVAB]UCAUGCCUUUCUACAGUGGCU 985 AGCCACUGUAGAAAGGCAUGAUU 986 D-1410 UCAUGCCUUUCUACAGUGGC{INVAB} 987 AGCCACUGUAGAAAGGCAUGAUU 988 D-1411 [INVAB]AUGCCUUUCUACAGUGGCUUU 989 AGCCACUGUAGAAAGGCAUUU 990 D-1412 UCAUGCCUUUCUACAGUGGC{INVAB} 991 AGCCACUGUAGAAAGGCAUGAUU 992 D-1413 UCAUGCCUUUCUACAGUGGC{INVAB} 993 AGCCACUGUAGAAAGGCAUGAUU 994 D-1414 [INVAB]UCAUGCCUUUCUACAGUGGC{INVAB} 995 AGCCACUGUAGAAAGGCAUGAUU 996 D-1415 [INVAB]AUGCCUUUCUACAGUGGCAUU 997 UGCCACUGUAGAAAGGCAUUU 998 D-1416 UCAUGCCUUUCUACAGUGGC{INVAB} 999 UGCCACUGUAGAAAGGCAUGAUU 1000 D-1417 [INVAB]UCAUGCCUUUCUACAGUGGCA 1001 UGCCACUGUAGAAAGGCAUGAUU 1002 D-1418 UCAUGCCUUUCUACAGUGGC{INVDA} 1003 UGCCACUGUAGAAAGGCAUGAUU 1004 D-1419 CUGCUUCAUGCCUUUCUACA{INVAB} 1005 AUGUAGAAAGGCAUGAAGCAGUU 1006 D-1420 CUGCUUCAUGCCUUUCUACA{INVAB} 1007 UUGUAGAAAGGCAUGAAGCAGUU 1008 D-1421 CUGCUUCAUGCCUUUCUACA{INVDA} 1009 UUGUAGAAAGGCAUGAAGCAGUU 1010 D-1422 UCAUGCCUUUCUACAGUGGC{INVAB} 1011 UGCCACUGUAGAAAGGCAUGAUU 1012 D-1423 UCAUGCCUUUCUACAGUGGC{INVDA} 1013 UGCCACUGUAGAAAGGCAUGAUU 1014 D-1424 [INVAB]CUUCAUGCCUUUCUACAGUUU 1015 ACUGUAGAAAGGCAUGAAGUU 1016 D-1425 CUGCUUCAUGCCUUUCUACA{INVAB} 1017 AUGUA[AB]AAAGGCAUGAAGCAGUU 1018 D-1426 CUGCUUCAUGCCUUUCUACA{INVAB} 1019 AUGUA[AB]AAAGGCAUGAAGCAGUU 1020 D-1427 UGCUUCAUGCCUUUCUACAG{INVAB} 1021 ACUGU[AB]GAAAGGCAUGAAGCAUU 1022 D-1428 UGCUUCAUGCCUUUCUACAG{INVAB} 1023 ACUGU[AB]GAAAGGCAUGAAGCAUU 1024 D-1429 UCAUGCCUUUCUACAGUGGC{INVAB} 1025 AGCCA[AB]UGUAGAAAGGCAUGAUU 1026 D-1430 UCAUGCCUUUCUACAGUGGC{INVAB} 1027 AGCCA[AB]UGUAGAAAGGCAUGAUU 1028 D-1431 CUGCUUCAUGCCUUUCUACA{INVAB} 1029 AU[GNA-G]UAGAAAGGCAUGAAGCAGUU 1030 D-1432 CUGCUUCAUGCCUUUCUACA{INVAB} 1031 AUG[GNA-U]AGAAAGGCAUGAAGCAGUU 1032 D-1433 CUGCUUCAUGCCUUUCUACA{INVAB} 1033 AUGU[GNA-A]GAAAGGCAUGAAGCAGUU 1034 D-1434 CUGCUUCAUGCCUUUCUACA{INVAB} 1035 AUGUA[GNA-G]AAAGGCAUGAAGCAGUU 1036 D-1435 CUGCUUCAUGCCUUUCUACA{INVAB} 1037 AUGUAG[GNA-A]AAGGCAUGAAGCAGUU 1038 D-1436 CUGCUUCAUGCCUUUCUACA{INVAB} 1039 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1040 D-1437 CUGCUUCAUGCCUUUCUACA{INVAB} 1041 AUGUAGAAAGGCAUGAAGCAGUU 1042 D-1438 [INVAB]CUGCUUCAUGCCUUUCUACAU 1043 AUGUAGAAAGGCAUGAAGCAGUU 1044 D-1439 [INVAB]CUGCUUCAUGCCUUUCUACA{INVAB} 1045 AUGUAGAAAGGCAUGAAGCAGUU 1046 D-1440 CUGCUUCAUGCCUUUCUACA{INVAB} 1047 AUGUAGAAAGGCAUGAAGCAGUU 1048 D-1441 CUGCUUCAUGCCUUUCUACA{INVAB} 1049 AUGUAGAAAGGCAUGAAGCAGUU 1050 D-1442 [INVAB]CUGCUUCAUGCCUUUCUACAU 1051 AUGUAGAAAGGCAUGAAGCAGUU 1052 D-1443 CUGCUUCAUGCCUUUCUACA{INVAB} 1053 AUGUAGAAAGGCAUGAAGCAGUU 1054 D-1444 CUGCUUCAUGCCUUUCUACA{INVAB} 1055 AUGUAGAAAGGCAUGAAGCAGUU 1056 D-1445 [INVAB]CUGCUUCAUGCCUUUCUACAU 1057 AUGUAGAAAGGCAUGAAGCAGUU 1058 D-1446 [INVAB]CUGCUUCAUGCCUUUCUACA{INVAB} 1059 AUGUAGAAAGGCAUGAAGCAGUU 1060 D-1447 CUGCUUCAUGCCUUUCUACA{INVAB} 1061 A[AB]GUAGAAAGGCAUGAAGCAGUU 1062 D-1448 CUGCUUCAUGCCUUUCUACA{INVAB} 1063 AU[AB]UAGAAAGGCAUGAAGCAGUU 1064 D-1449 CUGCUUCAUGCCUUUCUACA{INVAB} 1065 AUG[AB]AGAAAGGCAUGAAGCAGUU 1066 D-1450 CUGCUUCAUGCCUUUCUACA{INVAB} 1067 AUGU[AB]GAAAGGCAUGAAGCAGUU 1068 D-1451 CUGCUUCAUGCCUUUCUACA{INVAB} 1069 AUGUAG[AB]AAGGCAUGAAGCAGUU 1070 D-1452 CUGCUUCAUGCCUUUCUACA{INVAB} 1071 AUGUAGA[AB]AGGCAUGAAGCAGUU 1072 D-1453 CAACGUACCCUUCAUUGAUG{INVAB} 1073 ACAUCAAUGAAGGGUACGUUGUU 1074 D-1454 CAACGUACCCUUCAUUGAUG{INVAB} 1075 ACAUCAAUGAAGGGUACGUUGUU 1076 D-1455 ACAUGGCUUCCAGAUAUGCC{INVAB} 1077 AGGCAUAUCUGGAAGCCAUGUUU 1078 D-1456 ACAUGGCUUCCAGAUAUGCC{INVAB} 1079 AGGCAUAUCUGGAAGCCAUGUUU 1080 D-1457 CUGCUUCAUGCCUUUCUACA{INVAB} 1081 AUGUAGAAAGGCAUGAAGCAGUU 1082 D-1458 CUGCUUCAUGCCUUUCUACA{INVAB} 1083 AUGUAGAAAGGCAUGAAGCAGUU 1084 D-1459 CUGCUUCAUGCCUUUCUACA{INVAB} 1085 AUGUAGAAAGGCAUGAAGCAGUU 1086 D-1460 CUGCGGCUUCCUGGGCUUCU{INVAB} 1087 UAGAAGCCCAGGAAGCCGCAGUU 1088 D-1461 CUGCGGCUUCCUGGGCUUCU{INVAB} 1089 UAGAAGCCCAGGAAGCCGCAGUU 1090 D-1462 UGCUUCAUGCCUUUCUACAG{INVAB} 1091 ACUGUAGAAAGGCAUGAAGCAUU 1092 D-1463 [INVAB]UGCUUCAUGCCUUUCUACAGU 1093 ACUGUAGAAAGGCAUGAAGCAUU 1094 D-1464 [INVAB]UGCUUCAUGCCUUUCUACAG{INVAB} 1095 ACUGUAGAAAGGCAUGAAGCAUU 1096 D-1465 UGCUUCAUGCCUUUCUACAG{INVAB} 1097 ACUGUAGAAAGGCAUGAAGCAUU 1098 D-1466 UGCUUCAUGCCUUUCUACAG{INVAB} 1099 ACUGUAGAAAGGCAUGAAGCAUU 1100 D-1467 UGCUUCAUGCCUUUCUACAG{INVAB} 1101 ACUGUAGAAAGGCAUGAAGCAUU 1102 D-1468 CUGCUUCAUGCCUUUCUACAU 1103 AUGUAGAAAGGCAUGAAGCAGUU 1104 D-1469 CUGCUUCAUGCCUUUCUACAU 1105 AUGUAGAAAGGCAUGAAGCAGUU 1106 D-1470 CUGCUUCAUGCCUUUCUACAU 1107 AUGUAGAAAGGCAUGAAGCAGUU 1108 D-1471 UGCUUCAUGCCUUUCUACAG{INVAB} 1109 UCUGUAGAAAGGCAUGAAGCAUU 1110 D-1472 UGCUUCAUGCCUUUCUACAG{INVDA} 1111 UCUGUAGAAAGGCAUGAAGCAUU 1112 D-1473 CUGCUUCAUGCCUUUCUACA{INVDT} 1113 AUGUAGAAAGGCAUGAAGCAGUU 1114 D-1474 CUGCUUCAUGCCUUUCUACA{INVAB} 1115 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1116 D-1475 CUGCUUCAUGCCUUUCUACA{INVAB} 1117 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1118 D-1476 CUGCUUCAUGCCUUUCUACA{INVAB} 1119 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1120 D-1477 CUGCUUCAUGCCUUUCUACA{INVAB} 1121 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1122 D-1478 CUGCUUCAUGCCUUUCUACA{INVAB} 1123 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1124 D-1479 CUGCUUCAUGCCUUUCUACA{INVAB} 1125 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1126 D-1480 CUGCUUCAUGCCUUUCUACA{INVAB} 1127 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1128 D-1481 CUGCUUCAUGCCUUUCUACA{INVAB} 1129 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1130 D-1482 CUGCUUCAUGCCUUUCUACA{INVAB} 1131 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1132 D-1483 CUGCUUCAUGCCUUUCUACA{INVAB} 1133 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1134 D-1484 CUGCUUCAUGCCUUUCUACA{INVAB} 1135 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1136 D-1485 CUGCUUCAUGCCUUUCUACA{INVAB} 1137 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1138 D-1486 CUGCUUCAUGCCUUUCUACA{INVAB} 1139 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1140 D-1487 CUGCUUCAUGCCUUUCUACA{INVAB} 1141 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1142 D-1488 CUGCUUCAUGCCUUUCUACA{INVAB} 1143 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1144 D-1489 CUGCUUCAUGCCUUUCUACA{INVAB} 1145 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1146 D-1490 CUGCUUCAUGCCUUUCUACA{INVAB} 1147 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1148 D-1491 CUGCUUCAUGCCUUUCUACA{INVAB} 1149 AUGUAGA[GNA-A]A[DG]GCAUGAAGCAGUU 1150 D-1492 CUGCUUCAUGCCUUUCUACA{INVAB} 1151 AUGUAGA[GNA-A][DA]GGCAUGAAGCAGUU 1152 D-1493 CUGCUUCAUGCCUUUCUACA{INVAB} 1153 AUGUAGA[GNA-A]AG[DG]CAUGAAGCAGUU 1154 D-1494 CUGCUUCAUGCCUUUCUACA{INVAB} 1155 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1156 D-1495 CUGCUUCAUGCCUUUCUACA{INVAB} 1157 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1158 D-1496 CUGCUUCAUGCCUUUCUACA{INVAB} 1159 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1160 D-1497 CUGCUUCAUGCCUUUCUACA{INVAB} 1161 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1162 D-1498 CUGCUUCAUGCCUUUCUACA{INVAB} 1163 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1164 D-1499 CUGCUUCAUGCCUUUCUACA{INVAB} 1165 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1166 D-1500 CUGCUUCAUGCCUUUCUACA{INVAB} 1167 AUGUAGAAAGGCAUGAAGCAGUU 1168 D-1501 UGCUUCAUGCCUUUCUACAG{INVDA} 1169 U[GNA-C]UGUAGAAAGGCAUGAAGCAUU 1170 D-1502 UGCUUCAUGCCUUUCUACAG{INVDA} 1171 UC[GNA-U]GUAGAAAGGCAUGAAGCAUU 1172 D-1503 UGCUUCAUGCCUUUCUACAG{INVDA} 1173 UCUG[GNA-U]AGAAAGGCAUGAAGCAUU 1174 D-1504 UGCUUCAUGCCUUUCUACAG{INVDA} 1175 UCUGU[GNA-A]GAAAGGCAUGAAGCAUU 1176 D-1505 UGCUUCAUGCCUUUCUACAG{INVDA} 1177 UCUGUAG[GNA-A]AAGGCAUGAAGCAUU 1178 D-1506 UGCUUCAUGCCUUUCUACAG{INVDA} 1179 U[AB]UGUAGAAAGGCAUGAAGCAUU 1180 D-1507 UGCUUCAUGCCUUUCUACAG{INVDA} 1181 UC[AB]GUAGAAAGGCAUGAAGCAUU 1182 D-1508 UGCUUCAUGCCUUUCUACAG{INVDA} 1183 UCU[AB]UAGAAAGGCAUGAAGCAUU 1184 D-1509 UGCUUCAUGCCUUUCUACAG{INVDA} 1185 UCUG[AB]AGAAAGGCAUGAAGCAUU 1186 D-1510 UGCUUCAUGCCUUUCUACAG{INVDA} 1187 UCUGU[AB]GAAAGGCAUGAAGCAUU 1188 D-1511 UGCUUCAUGCCUUUCUACAG{INVDA} 1189 UCUGUA[AB]AAAGGCAUGAAGCAUU 1190 D-1512 UGCUUCAUGCCUUUCUACAG{INVDA} 1191 UCUGUAG[AB]AAGGCAUGAAGCAUU 1192 D-1513 UCAUGCCUUUCUACAGUGGC{INVDT} 1193 AGCCACUGUAGAAAGGCAUGAUU 1194 D-1514 UCAUGCCUUUCUACAGUGGC{INVAB} 1195 UGCCACUGUAGAAAGGCAUGAUU 1196 D-1515 UCAUGCCUUUCUACAGUGGC{INVDA} 1197 UGCCACUGUAGAAAGGCAUGAUU 1198 D-1516 UCCUGCUUCAUGCCUUUCUA{INVDT} 1199 AUAGAAAGGCAUGAAGCAGGAUU 1200 D-1517 UCCUGCUUCAUGCCUUUCUA{INVAB} 1201 UUAGAAAGGCAUGAAGCAGGAUU 1202 D-1518 UCCUGCUUCAUGCCUUUCUA{INVDA} 1203 UUAGAAAGGCAUGAAGCAGGAUU 1204 D-1519 UAUGUUCCUGCUUCAUGCCU{INVDT} 1205 AAGGCAUGAAGCAGGAACAUAUU 1206 D-1520 UAUGUUCCUGCUUCAUGCCU{INVAB} 1207 UAGGCAUGAAGCAGGAACAUAUU 1208 D-1521 UAUGUUCCUGCUUCAUGCCU{INVDA} 1209 UAGGCAUGAAGCAGGAACAUAUU 1210 D-1522 UCCUGCUUCAUGCCUUUCUA{INVAB} 1211 AUAGAAAGGCAUGAAGCAGGAUU 1212 D-1523 UAUGUUCCUGCUUCAUGCCU{INVAB} 1213 AAGGCAUGAAGCAGGAACAUAUU 1214 D-1524 UCAUGCCUUUCUACAGUGGC{INVDT} 1215 AGCCACUGUAGAAAGGCAUGAUU 1216 D-1525 UCCUGCUUCAUGCCUUUCUA{INVDT} 1217 AUAGAAAGGCAUGAAGCAGGAUU 1218 D-1526 UCCUGCUUCAUGCCUUUCUA{INVDA} 1219 UUAGAAAGGCAUGAAGCAGGAUU 1220 D-1527 UAUGUUCCUGCUUCAUGCCU{INVDA} 1221 UAGGCAUGAAGCAGGAACAUAUU 1222 D-1528 UGCUUCAUGCCUUUCUACAG{INVDA} 1223 UCUGUA[GNA-G]AAAGGCAUGAAGCAUU 1224 D-1529 CUGCUUCAUGCCUUUCUACA{INVAB} 1225 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1226 D-1530 CUGCUUCAUGCCUUUCUACA{INVAB} 1227 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1228 D-1531 CUGCUUCAUGCCUUUCUACA{INVAB} 1229 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1230 D-1532 CUGCUUCAUGCCUUUCUACA{INVAB} 1231 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1232 D-1533 CUGCUUCAUGCCUUUCUACA{INVAB} 1233 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1234 D-1534 CUGCUUCAUGCCUUUCUACA{INVAB} 1235 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1236 D-1535 CUGCUUCAUGCCUUUCUACA{INVAB} 1237 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1238 D-1536 CUGCUUCAUGCCUUUCUACA{INVAB} 1239 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1240 D-1537 CUGCUUCAUGCCUUUCUACA{INVAB} 1241 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1242 D-1538 CUGCUUCAUGCCU[LNA- 1243 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1244 T]UCUACA{INVAB} D-1539 CUGCUUCAUGCC[LNA- 1245 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1246 T]UUCUACA{INVAB} D-1540 CUGCUUCAUGCCUU[LNA- 1247 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1248 T]CUACA{INVAB} D-1541 CUGCUUCAUGCCUUUC[LNA- 1249 AUGU[GNA-A]GAAAGGCAUGAAGCAGUU 1250 T]ACA{INVAB} D-1542 CUGCUUCAUGCCUUUCU[LNA- 1251 AUG[GNA-U]AGAAAGGCAUGAAGCAGUU 1252 A]CA{INVAB} D-1543 CUGCUUCAUGCCUUUCUAC[LNA- 1253 A[GNA-U]GUAGAAAGGCAUGAAGCAGUU 1254 A]{INVAB} D-1544 CUGCUUCAUGCCUU[LNA- 1255 AUGUAG[AB]AAGGCAUGAAGCAGUU 1256 T]CUACA{INVAB} D-1545 CUGCUUCAUGCCUUUC[LNA- 1257 AUGU[B]GAAAGGCAUGAAGCAGUU 1258 T]ACA{INVAB} D-1546 CUGCUUCAUGCCUUUCU[LNA- 1259 AUG[AB]AGAAAGGCAUGAAGCAGUU 1260 A]CA{INVAB} D-1547 CUGCUUCAUGCCUUUCUACA{INVAB} 1261 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1262 D-1548 CUGCUUCAUGCCUUUCUACA{INVAB} 1263 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1264 D-1549 CUGCUUCAUGCCU[LNA- 1265 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1266 T]UCUACA{INVAB} D-1550 CUGCUUCAUGCCU[LNA- 1267 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1268 T]UCUACA{INVAB} D-1551 CUGCUUCAUGCCU[LNA- 1269 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1270 T]UCUACA{INVAB} D-1552 CUGCUUCAUGCCU[LNA- 1271 AUGUAGA[GNA-A]AGGCAUGAAGCAGUU 1272 T]UCUACA{INVAB} D-1553 UGCUUCAUGCCUUUCU[LNA- 1273 UCUG[AB]AGAAAGGCAUGAAGCAUU 1274 A]CAG{INVDA} D-1554 UGCUUCAUGCCUUUC[LNA- 1275 UCUGU[AB]GAAAGGCAUGAAGCAUU 1276 T]ACAG{INVDA} D-1555 UGCUUCAUGCCUU[LNA- 1277 UCUGUAG[AB]AAGGCAUGAAGCAUU 1278 T]CUACAG{INVDA} D-1556 UGCUUCAUGCCUUUCUAC[LNA- 1279 UC[GNA-U]GUAGAAAGGCAUGAAGCAUU 1280 A]G{INVDA} D-1557 UGCUUCAUGCCUUUCU[LNA- 1281 UCUG[GNA-U]AGAAAGGCAUGAAGCAUU 1282 A]CAG{INVDA} D-1558 UGCUUCAUGCCUUUC[LNA- 1283 UCUGU[GNA-A]GAAAGGCAUGAAGCAUU 1284 T]ACAG{INVDA} D-1559 UGCUUCAUGCCUUUCUACA[LNA- 1285 U[AB]UGUAGAAAGGCAUGAAGCAUU 1286 G]{INVDA} D-1560 UGCUUCAUGCCUUUCUAC[LNA- 1287 UC[AB]GUAGAAAGGCAUGAAGCAUU 1288 A]G{INVDA} D-1561 UCAUGCCUUUCUACA[LNA- 1289 AGCCA[AB]UGUAGAAAGGCAUGAUU 1290 G]UGGC{INVAB} D-1562 UCAUGCCUUUCUACAG[LNA- 1291 AGCCA[AB]UGUAGAAAGGCAUGAUU 1292 T]GGC{INVAB} D-1563 UCAUGCCUUUCUACAGUGGC{INVAB} 1293 AGCCA[AB]UGUAGAAAGGCAUGAUU 1294 D-1564 GGUAUGUUCCUGCUUCAUUUU 2257 AAUGAAGCAGGAACAUACCUU 2258 D-1565 GGUAUGUUCCUGCUUCAUAUU 2259 UAUGAAGCAGGAACAUACCUU 2260 D-1566 GUAUGUUCCUGCUUCAUGUUU 2261 ACAUGAAGCAGGAACAUACUU 2262 D-1567 UAUGUUCCUGCUUCAUGCAUU 2263 UGCAUGAAGCAGGAACAUAUU 2264 D-1568 AUGUUCCUGCUUCAUGCCUUU 2265 AGGCAUGAAGCAGGAACAUUU 2266 D-1569 UGUUCCUGCUUCAUGCCUUUU 2267 AAGGCAUGAAGCAGGAACAUU 2268 D-1570 GUUCCUGCUUCAUGCCUUUUU 2269 AAAGGCAUGAAGCAGGAACUU 2270 D-1571 UUCCUGCUUCAUGCCUUUUUU 2271 AAAAGGCAUGAAGCAGGAAUU 2272 D-1572 UUCCUGCUUCAUGCCUUUAUU 2273 UAAAGGCAUGAAGCAGGAAUU 2274 D-1573 UCCUGCUUCAUGCCUUUCUUU 2275 AGAAAGGCAUGAAGCAGGAUU 2276 D-1574 CCUGCUUCAUGCCUUUCUAUU 2277 UAGAAAGGCAUGAAGCAGGUU 2278 D-1575 CUGCUUCAUGCCUUUCUAUUU 2279 AUAGAAAGGCAUGAAGCAGUU 2280 D-1576 CUGCUUCAUGCCUUUCUAAUU 2281 UUAGAAAGGCAUGAAGCAGUU 2282 D-1577 UGCUUCAUGCCUUUCUACAUU 2283 UGUAGAAAGGCAUGAAGCAUU 2284 D-1578 GCUUCAUGCCUUUCUACAUUU 2285 AUGUAGAAAGGCAUGAAGCUU 2286 D-1579 GCUUCAUGCCUUUCUACAAUU 2287 UUGUAGAAAGGCAUGAAGCUU 2288 D-1580 CUUCAUGCCUUUCUACAGUUU 2289 ACUGUAGAAAGGCAUGAAGUU 2290 D-1581 UUCAUGCCUUUCUACAGUUUU 2291 AACUGUAGAAAGGCAUGAAUU 2292 D-1582 UUCAUGCCUUUCUACAGUAUU 2293 UACUGUAGAAAGGCAUGAAUU 2294 D-1583 UCAUGCCUUUCUACAGUGUUU 2295 ACACUGUAGAAAGGCAUGAUU 2296 D-1584 UCAUGCCUUUCUACAGUGAUU 2297 UCACUGUAGAAAGGCAUGAUU 2298 D-1585 CAUGCCUUUCUACAGUGGUUU 2299 ACCACUGUAGAAAGGCAUGUU 2300 D-1586 CAUGCCUUUCUACAGUGGAUU 2301 UCCACUGUAGAAAGGCAUGUU 2302 D-1587 AUGCCUUUCUACAGUGGCUUU 2303 AGCCACUGUAGAAAGGCAUUU 2304 D-1588 AUGCCUUUCUACAGUGGCAUU 2305 UGCCACUGUAGAAAGGCAUUU 2306 D-1589 UGCCUUUCUACAGUGGCCUUU 2307 AGGCCACUGUAGAAAGGCAUU 2308 D-1590 GCCUUUCUACAGUGGCCUUUU 2309 AAGGCCACUGUAGAAAGGCUU 2310 D-1591 GGUAUGUUCCUGCUUCAUCUU 2311 GAUGAAGCAGGAACAUACCUU 2312 D-1592 GUAUGUUCCUGCUUCAUCCUU 2313 GGAUGAAGCAGGAACAUACUU 2314 D-1593 UAUGUUCCUGCUUCAUCCCUU 2315 GGGAUGAAGCAGGAACAUAUU 2316 D-1594 AUGUUCCUGCUUCAUCCCCUU 2317 GGGGAUGAAGCAGGAACAUUU 2318 D-1595 UGUUCCUGCUUCAUCCCCUUU 2319 AGGGGAUGAAGCAGGAACAUU 2320 D-1596 GUUCCUGCUUCAUCCCCUUUU 2321 AAGGGGAUGAAGCAGGAACUU 2322 D-1597 UUCCUGCUUCAUCCCCUUCUU 2323 GAAGGGGAUGAAGCAGGAAUU 2324 D-1598 UCCUGCUUCAUCCCCUUCUUU 2325 AGAAGGGGAUGAAGCAGGAUU 2326 D-1599 CCUGCUUCAUCCCCUUCUAUU 2327 UAGAAGGGGAUGAAGCAGGUU 2328 D-1600 CUGCUUCAUCCCCUUCUACUU 2329 GUAGAAGGGGAUGAAGCAGUU 2330 D-1601 UGCUUCAUCCCCUUCUACAUU 2331 UGUAGAAGGGGAUGAAGCAUU 2332 D-1602 GCUUCAUCCCCUUCUACAGUU 2333 CUGUAGAAGGGGAUGAAGCUU 2334 D-1603 CUUCAUCCCCUUCUACAGUUU 2335 ACUGUAGAAGGGGAUGAAGUU 2336 D-1604 UUCAUCCCCUUCUACAGUGUU 2337 CACUGUAGAAGGGGAUGAAUU 2338 D-1605 UCAUCCCCUUCUACAGUGGUU 2339 CCACUGUAGAAGGGGAUGAUU 2340 D-1606 CAUCCCCUUCUACAGUGGCUU 2341 GCCACUGUAGAAGGGGAUGUU 2342 D-1607 AUCCCCUUCUACAGUGGCCUU 2343 GGCCACUGUAGAAGGGGAUUU 2344 D-1608 UCCCCUUCUACAGUGGCCUUU 2345 AGGCCACUGUAGAAGGGGAUU 2346 D-1609 CCCCUUCUACAGUGGCCUUUU 2347 AAGGCCACUGUAGAAGGGGUU 2348

To improve the potency and in vivo stability of PNPLA3 siRNA sequences, chemical modifications were incorporated into PNPLA3 siRNA molecules. Specifically, 2′-O-methyl and 2′-fluoro modifications of the ribose sugar were incorporated at specific positions within the PNPLA3 siRNAs. Phosphorothioate internucleotide linkages were also incorporated at the terminal ends of the antisense and/or sense sequences. Table 2 below depicts the modifications in the sense and antisense sequences for each of the modified PNPLA3 siRNAs. The nucleotide sequences in Table X are listed according to the following notations: A, U, G, and C=corresponding ribonucleotide; dT=deoxythymidine; a, u, g, and c=corresponding 2′-O-methyl ribonucleotide; Af, Uf, Gf, and Cf=corresponding 2′-deoxy-2′-fluoro (“2′-fluoro”) ribonucleotide. Insertion of an “s” in the sequence indicates that the two adjacent nucleotides are connected by a phosphorothiodiester group (e.g. a phosphorothioate internucleotide linkage). Unless indicated otherwise, all other nucleotides are connected by 3′-5′ phosphodiester groups. Each of the siRNA compounds in Table 2 comprises a 19 base pair duplex region with either a 2 nucleotide overhang at the 3′ end of both strands or bluntmer at one or both ends. The GalNAc3K2AhxC6 is:

TABLE 2 siRNA sequences directed to PNPLA3 with modifications SEQ ID SEQ ID Duplex NO: NO: No. Sense sequence (5′-3′) (sense) Antisense sequence (5′-3′) (antisense) D-2000 GfsgsGfcAfaUfaAfAfGfuAfcCfuGfcUf 167 asGfscAfgGfuAfcuuUfaUfuGfcCfcsUfs 168 susUf u D-2001 CfsgsGfcCfaAfuGfUfCfcAfcCfaGfcUf 169 asGfscUfgGfuGfgacAfuUfgGfcCfgsUfs 170 susUf u D-2002 GfsgsUfcCfaGfcCfUfGfaAfcUfuCfuUf 171 asAfsgAfaGfuUfcagGfcUfgGfaCfcsUfs 172 susUf u D-2003 GfscsUfuCfaUfcCfCfCfuUfcUfaCfaGf 173 csUfsgUfaGfaAfgggGfaUfgAfaGfcsUfs 174 susUf u D-2004 GfscsGfgCfuUfcCfUfGfgGfcUfuCfuAf 175 usAfsgAfaGfcCfcagGfaAfgCfcGfcsUfs 176 susUf u D-2005 GfscsCfuCfuGfaGfCfUfgAfgUfuGfgUf 177 asCfscAfaCfuCfagcUfcAfgAfgGfcsUfs 178 susUf u D-2006 GfsusGfaCfaAfcGfUfAfcCfcUfuCfaUf 179 asUfsgAfaGfgGfuacGfuUfgUfcAfcsUfs 180 susUf u D-2007 CfscsCfgCfcUfcCfAfGfgUfcCfcAfaAf 181 usUfsuGfgGfaCfcugGfaGfgCfgGfgsUfs 182 susUf u D-2008 CfsusUfcAfuCfcCfCfUfuCfuAfcAfgUf 183 asCfsuGfuAfgAfaggGfgAfuGfaAfgsUfs 184 susUf u D-2009 GfsgsUfaUfgUfuCfCfUfgCfuUfcAfuGf 185 csAfsuGfaAfgCfaggAfaCfaUfaCfcsUfs 186 susUf u D-2010 GfsusAfuGfuUfcCfUfGfcUfuCfaUfgCf 187 gsCfsaUfgAfaGfcagGfaAfcAfuAfcsUfs 188 susUf u D-2011 UfsasUfgUfuCfcUfGfCfuUfcAfuGfcCf 189 gsGfscAfuGfaAfgcaGfgAfaCfaUfasUfs 190 susUf u D-2012 AfsusGfuUfcCfuGfCfUfuCfaUfgCfcCf 191 gsGfsgCfaUfgAfagcAfgGfaAfcAfusUfs 192 susUf u D-2013 UfsgsUfuCfcUfgCfUfUfcAfuGfcCfcUf 193 asGfsgGfcAfuGfaagCfaGfgAfaCfasUfs 194 susUf u D-2014 GfsusUfcCfuGfcUfUfCfaUfgCfcCfuUf 195 asAfsgGfgCfaUfgaaGfcAfgGfaAfcsUfs 196 susUf u D-2015 UfsusCfcUfgCfuUfCfAfuGfcCfcUfuCf 197 gsAfsaGfgGfcAfugaAfgCfaGfgAfasUfs 198 susUf u D-2016 UfscsCfuGfcUfuCfAfUfgCfcCfuUfcUf 199 asGfsaAfgGfgCfaugAfaGfcAfgGfasUfs 200 susUf u D-2017 CfscsUfgCfuUfcAfUfGfcCfcUfuCfuAf 201 usAfsgAfaGfgGfcauGfaAfgCfaGfgsUfs 202 susUf u D-2018 CfsusGfcUfuCfaUfGfCfcCfuUfcUfaCf 203 gsUfsaGfaAfgGfgcaUfgAfaGfcAfgsUfs 204 susUf u D-2019 UfsgsCfuUfcAfuGfCfCfcUfuCfuAfcAf 205 usGfsuAfgAfaGfggcAfuGfaAfgCfasUfs 206 susUf u D-2020 GfscsUfuCfaUfgCfCfCfuUfcUfaCfaGf 207 csUfsgUfaGfaAfgggCfaUfgAfaGfcsUfs 208 susUf u D-2021 CfsusUfcAfuGfcCfCfUfuCfuAfcAfgUf 209 asCfsuGfuAfgAfaggGfcAfuGfaAfgsUfs 210 susUf u D-2022 UfsusCfaUfgCfcCfUfUfcUfaCfaGfuGf 211 csAfscUfgUfaGfaagGfgCfaUfgAfasUfs 212 susUf u D-2023 UfscsAfuGfcCfcUfUfCfuAfcAfgUfgGf 213 csCfsaCfuGfuAfgaaGfgGfcAfuGfasUfs 214 susUf u D-2024 CfsasUfgCfcCfuUfCfUfaCfaGfuGfgCf 215 gsCfscAfcUfgUfagaAfgGfgCfaUfgsUfs 216 susUf u D-2025 AfsusGfcCfcUfuCfUfAfcAfgUfgGfcCf 217 gsGfscCfaCfuGfuagAfaGfgGfcAfusUfs 218 susUf u D-2026 UfsgsCfcCfuUfcUfAfCfaGfuGfgCfcUf 219 asGfsgCfcAfcUfguaGfaAfgGfgCfasUfs 220 susUf u D-2027 GfscsCfcUfuCfuAfCfAfgUfgGfcCfuUf 221 asAfsgGfcCfaCfuguAfgAfaGfgGfcsUfs 222 susUf u D-2028 GfsgsUfaUfgUfuCfCfUfgCfuUfcAfuCf 223 gsAfsuGfaAfgCfaggAfaCfaUfaCfcsUfs 224 susUf u D-2029 GfsusAfuGfuUfcCfUfGfcUfuCfaUfcCf 225 gsGfsaUfgAfaGfcagGfaAfcAfuAfcsUfs 226 susUf u D-2030 UfsasUfgUfuCfcUfGfCfuUfcAfuCfcCf 227 gsGfsgAfuGfaAfgcaGfgAfaCfaUfasUfs 228 susUf u D-2031 AfsusGfuUfcCfuGfCfUfuCfaUfcCfcCf 229 gsGfsgGfaUfgAfagcAfgGfaAfcAfusUfs 230 susUf u D-2032 UfsgsUfuCfcUfgCfUfUfcAfuCfcCfcUf 231 asGfsgGfgAfuGfaagCfaGfgAfaCfasUfs 232 susUf u D-2033 GfsusUfcCfuGfcUfUfCfaUfcCfcCfuUf 233 asAfsgGfgGfaUfgaaGfcAfgGfaAfcsUfs 234 susUf u D-2034 UfsusCfcUfgCfuUfCfAfuCfcCfcUfuCf 235 gsAfsaGfgGfgAfugaAfgCfaGfgAfasUfs 236 susUf u D-2035 UfscsCfuGfcUfuCfAfUfcCfcCfuUfcUf 237 asGfsaAfgGfgGfaugAfaGfcAfgGfasUfs 238 susUf u D-2036 CfscsUfgCfuUfcAfUfCfcCfcUfuCfuAf 239 usAfsgAfaGfgGfgauGfaAfgCfaGfgsUfs 240 susUf u D-2037 CfsusGfcUfuCfaUfCfCfcCfuUfcUfaCf 241 gsUfsaGfaAfgGfggaUfgAfaGfcAfgsUfs 242 susUf u D-2038 UfsgsCfuUfcAfuCfCfCfcUfuCfuAfcAf 243 usGfsuAfgAfaGfgggAfuGfaAfgCfasUfs 244 susUf u D-2039 UfsusCfaUfcCfcCfUfUfcUfaCfaGfuGf 245 csAfscUfgUfaGfaagGfgGfaUfgAfasUfs 246 susUf u D-2040 UfscsAfuCfcCfcUfUfCfuAfcAfgUfgGf 247 csCfsaCfuGfuAfgaaGfgGfgAfuGfasUfs 248 susUf u D-2041 CfsasUfcCfcCfuUfCfUfaCfaGfuGfgCf 249 gsCfscAfcUfgUfagaAfgGfgGfaUfgsUfs 250 susUf u D-2042 UfscsCfcCfuUfcUfAfCfaGfuGfgCfcUf 251 asGfsgCfcAfcUfguaGfaAfgGfgGfasUfs 252 susUf u D-2043 GfsasUfcAfgGfaCfCfCfgAfgCfcGfaUf 253 asUfscGfgCfuCfgggUfcCfuGfaUfcsUfs 254 susUf u D-2044 UfsgsGfgCfuUfcUfAfCfcAfcGfuCfgUf 255 asCfsgAfcGfuGfguaGfaAfgCfcCfasUfs 256 susUf u D-2045 GfsasGfcGfaGfcAfCfGfcCfcCfgCfaUf 257 asUfsgCfgGfgGfcguGfcUfcGfcUfcsUfs 258 susUf u D-2046 UfsgsCfaCfuGfcGfUfCfgGfcGfuCfcUf 259 asGfsgAfcGfcCfgacGfcAfgUfgCfasUfs 260 susUf u D-2047 UfsgsGfaGfcAfgAfCfUfcUfgCfaGfgUf 261 asCfscUfgCfaGfaguCfuGfcUfcCfasUfs 262 susUf D-2048 UfsgsCfaGfgUfcCfUfCfuCfaGfaUfcUf 263 asGfsaUfcUfgAfgagGfaCfcUfgCfasUfs 264 susUf u D-2049 CfscsCfgGfcCfaAfUfGfuCfcAfcCfaUf 265 asUfsgGfuGfgAfcauUfgGfcCfgGfgsUfs 266 susUf u D-2050 UfsusCfuAfcAfgUfGfGfcCfuUfaUfcUf 267 asGfsaUfaAfgGfccaCfuGfuAfgAfasUfs 268 susUf u D-2051 UfscsUfaCfaGfuGfGfCfcUfuAfuCfcUf 269 asGfsgAfuAfaGfgccAfcUfgUfaGfasUfs 270 susUf u D-2052 CfsusUfcCfuUfcAfGfAfgGfcGfuGfcUf 271 asGfscAfcGfcCfucuGfaAfgGfaAfgsUfs 272 susUf u D-2053 UfsusCfcUfuCfaGfAfGfgCfgUfgCfgAf 273 usCfsgCfaCfgCfcucUfgAfaGfgAfasUfs 274 susUf u D-2054 GfscsGfuGfcGfaUfAfUfgUfgGfaUfgUf 275 asCfsaUfcCfaCfauaUfcGfcAfcGfcsUfs 276 susUf u D-2055 CfsgsUfgCfgAfuAfUfGfuGfgAfuGfgAf 277 usCfscAfuCfcAfcauAfuCfgCfaCfgsUfs 278 susUf u D-2056 UfsgsGfaUfgGfaGfGfAfgUfgAfgUfgAf 279 usCfsaCfuCfaCfuccUfcCfaUfcCfasUfs 280 susUf u D-2057 AfscsGfuAfcCfcUfUfCfaUfuGfaUfgUf 281 asCfsaUfcAfaUfgaaGfgGfuAfcGfusUfs 282 susUf u D-2058 UfsgsGfaCfaUfcAfCfCfaAfgCfuCfaUf 283 asUfsgAfgCfuUfgguGfaUfgUfcCfasUfs 284 susUf u D-2059 CfsasCfcUfgCfgUfCfUfcAfgCfaUfcUf 285 asGfsaUfgCfuGfagaCfgCfaGfgUfgsUfs 286 susUf u D-2060 AfscsCfuGfcGfuCfUfCfaGfcAfuCfcUf 287 asGfsgAfuGfcUfgagAfcGfcAfgGfusUfs 288 susUf u D-2061 CfscsAfgAfgAfcUfGfGfuGfaCfaUfgUf 289 asCfsaUfgUfcAfccaGfuCfuCfuGfgsUfs 290 susUf u D-2062 AfsusGfgCfuUfcCfAfGfaUfaUfgCfcUf 291 asGfsgCfaUfaUfcugGfaAfgCfcAfusUfs 292 susUf u D-2063 CfscsGfcCfuCfcAfGfGfuCfcCfaAfaUf 293 asUfsuUfgGfgAfccuGfgAfgGfcGfgsUfs 294 susUf u D-2064 UfsasCfcUfgCfuGfGfUfgCfuGfaGfgUf 295 asCfscUfcAfgCfaccAfgCfaGfgUfasUfs 296 susUf u D-2065 AfscsCfuGfcUfgGfUfGfcUfgAfgGfgUf 297 asCfscCfuCfaGfcacCfaGfcAfgGfusUfs 298 susUf u D-2066 CfsusCfuCfcAfcCfUfUfuCfcCfaGfuUf 299 asAfscUfgGfgAfaagGfuGfgAfgAfgsUfs 300 susUf u D-2067 UfsusUfuUfcAfcCfUfAfaCfuAfaAfaUf 301 asUfsuUfuAfgUfuagGfuGfaAfaAfasUfs 302 susUf u D-2068 CfgGfcCfaAfuGfUfCfcAfcCfaGfcUfsu 303 asGfscUfgGfuGfgacAfuUfgGfcCfgsUfs 304 sUf u D-2069 GfgUfcCfaGfcCfUfGfaAfcUfuCfuUfsu 305 asAfsgAfaGfuUfcagGfcUfgGfaCfcsUfs 306 sUf u D-2070 GfcGfgCfuUfcCfUfGfgGfcUfuCfuAfsu 307 usAfsgAfaGfcCfcagGfaAfgCfcGfcsUfs 308 sUf u D-2071 GfuGfaCfaAfcGfUfAfcCfcUfuCfaUfsu 309 asUfsgAfaGfgGfuacGfuUfgUfcAfcsUfs 310 sUf u D-2072 GfgUfaUfgUfuCfCfUfgCfuUfcAfuGfsu 311 csAfsuGfaAfgCfaggAfaCfaUfaCfcsUfs 312 sUf u D-2073 GfuAfuGfuUfcCfUfGfcUfuCfaUfgCfsu 313 gsCfsaUfgAfaGfcagGfaAfcAfuAfcsUfs 314 sUf u D-2074 UfgUfuCfcUfgCfUfUfcAfuGfcCfcUfsu 315 asGfsgGfcAfuGfaagCfaGfgAfaCfasUfs 316 sUf u D-2075 GfuUfcCfuGfcUfUfCfaUfgCfcCfuUfsu 317 asAfsgGfgCfaUfgaaGfcAfgGfaAfcsUfs 318 sUf u D-2076 CfcUfgCfuUfcAfUfGfcCfcUfuCfuAfsu 319 usAfsgAfaGfgGfcauGfaAfgCfaGfgsUfs 320 sUf u D-2077 GfcUfuCfaUfgCfCfCfuUfcUfaCfaGfsu 321 csUfsgUfaGfaAfgggCfaUfgAfaGfcsUfs 322 sUf u D-2078 CfuUfcAfuGfcCfCfUfuCfuAfcAfgUfsu 323 asCfsuGfuAfgAfaggGfcAfuGfaAfgsUfs 324 sUf u D-2079 UfuCfaUfgCfcCfUfUfcUfaCfaGfuGfsu 325 csAfscUfgUfaGfaagGfgCfaUfgAfasUfs 326 sUf u D-2080 AfuGfgCfuUfcCfAfGfaUfaUfgCfcUfsu 327 asGfsgCfaUfaUfcugGfaAfgCfcAfusUfs 328 sUf u D-2081 AfuGfcCfcUfuCfUfAfcAfgUfgGfcCfsu 329 gsGfscCfaCfuGfuagAfaGfgGfcAfusUfs 330 sUf u D-2082 GfcUfuCfaUfgCfCfCfuUfcUfaCfaUfsu 331 asUfsgUfaGfaAfgggCfaUfgAfaGfcsUfs 332 sUf u D-2083 {Phosphate}GfsgsAfaAfgAfcUfGfUfu 1295 {Phosphate}usUfsuUfuGfgAfacaGfuCf 1296 CfcAfoAfaAfsusUf uUfuCfcsUfsu D-2084 {GalNAc3K2AhxC6}ggsuaugUfuCfCfUf 1297 {Phosphate}csAfsuGfaAfGfcaggAfaCf 1298 Gfcuucaugsusu auaccsusu D-2085 {GalNAc3K2AhxC6}guauguUfcCfUfGfC 1299 {Phosphate}gsCfsaUfgAfAfgcagGfaAf 1300 fuucaugcsusu cauacsusu D-2086 {GalNAc3K2AhxC6}uguuccUfgCfUfUfC 1301 {Phosphate}asGfsgGfcAfUfgaagCfaGf 1302 faugcccususu gaacasusu D-2087 {GalNAc3K2AhxC6}gcuucaUfgCfCfCfU 1303 {Phosphate}csUfsgUfaGfAfagggCfaUf 1304 fucuacagsusu gaagcsusu D-2088 {GalNAc3K2AhxC6}cuucauGfcCfCfUfU 1305 {Phosphate}asCfsuGfuAfGfaaggGfcAf 1306 fcuacagususu ugaagsusu D-2089 {GalNAc3K2AhxC6}gcggcuUfcCfUfGfG 1307 {Phosphate}usAfsgAfaGfCfccagGfaAf 1308 fgcuucuasusu gccgcsusu D-2090 {GalNAc3K2AhxC6}guuccuGfcUfUfCfA 1309 {Phosphate}asAfsgGfgCfAfugaaGfcAf 1310 fugcccuususu ggaacsusu D-2091 {GalNAc3K2AhxC6}AfuGfgCfuUfcCfAf 1311 {Phosphate}asGfsgCfaUfaUfcugGfaAf 1312 GfaUfaUfgCfcUfsusUf gCfcAfusUfsu D-2092 {GalNAc3K2AhxC6}ccugcuUfcAfUfGfC 1313 {Phosphate}usAfsgAfaGfGfgcauGfaAf 1314 fccuucuasusu gcaggsusu D-2093 {GalNAc3K2AhxC6}uucaugCfcCfUfUfC 1315 {Phosphate}asAfscUfgUfAfgaagGfgCf 1316 fuacaguususu augaasusu D-2094 {GalNAc3K2AhxC6}uucaugCfcCfUfUfC 1317 {Phosphate}csAfscUfgUfAfgaagGfgCf 1318 fuacagugsusu augaasusu D-2095 {GalNAc3K2AhxC6}gcuucaUfgCfCfCfU 1319 {Phosphate}asUfsgUfaGfAfagggCfaUf 1320 fucuacaususu gaagcsusu D-2096 {GalNAc3K2AhxC6}gguccaGfcCfUfGfA 1321 {Phosphate}asAfsgAfaGfUfucagGfcUf 1322 facuucuususu ggaccsusu D-2097 {GalNAc3K2AhxC6}GfcGfgCfuUfcCfUf 1323 {Phosphate}usAfsgAfaGfCfccagGfaAf 1324 GfGfgcUfuCfuAfsusUf gCfcGfcsUfsu D-2098 {GalNAc3K2AhxC6}GfcGfgCfuUfcCfuG 1325 {Phosphate}usAfsgAfaGfCfccAfgGfaA 1326 fGfgcUfuCfuAfsusUf fgCfcGfcsUfsu D-2099 {GalNAc3K2AhxC6}GfuUfcCfuGfcUfUf 1327 {Phosphate}asAfsgGfgCfAfugaaGfcAf 1328 CfAfugCfcCfuUfsusUf gGfaAfcsUfsu D-2100 {GalNAc3K2AhxC6}GfuUfcCfuGfcUfuC 1329 {Phosphate}asAfsgGfgCfAfugAfaGfcA 1330 fAfugCfcCfuUfsusUf fgGfaAfcsUfsu D-2101 {GalNAc3K2AhxC6}CfcUfgCfuUfcAfUf 1331 {Phosphate}usAfsgAfaGfGfgcauGfaAf 1332 GfCfccUfuCfuAfsusUf gCfaGfgsUfsu D-2102 {GalNAc3K2AhxC6}CfcUfgCfuUfcAfuG 1333 {Phosphate}usAfsgAfaGfGfgcAfuGfaA 1334 fCfccUfuCfuAfsusUf fgCfaGfgsUfsu D-2103 {GalNAc3K2AhxC6}augcccUfuCfUfAfC 1335 {Phosphate}gsGfscCfaCfUfguagAfaGf 1336 faguggccsusu ggcaususu D-2104 {GalNAc3K2AhxC6}auggcuUfcCfAfGfA 1337 {Phosphate}asGfsgCfaUfAfucugGfaAf 1338 fuaugccususu gccaususu D-2105 {GalNAc3K2AhxC6}gugacaAfcGfUfAfC 1339 {Phosphate}asUfsgAfaGfGfguacGfuUf 1340 fccuucaususu gucacsusu D-2106 {GalNAc3K2AhxC6}guauguUfcCfUfGfC 1341 {Phosphate}gsCfsaUfgAfAfgcAfgGfaA 1342 fuucaugcsusu fcauacsusu D-2107 {GalNAc3K2AhxC6}guauguUfcCfuGfCf 1343 {Phosphate}gsCfsaUfgAfAfgcagGfaAf 1344 uucaugcsusu cauacsusu D-2108 {GalNAc3K2AhxC6}guauguUfcCfuGfCf 1345 {Phosphate}gsCfsaUfgAfAfgcAfgGfaA 1346 uucaugcsusu fcauacsusu D-2109 {GalNAc3K2AhxC6}guauguUfcCfUfGfC 1347 {Phosphate}asGfsgCfaUfgAfAfgcagGf 1348 fuucaugcscsu aAfcauacsusu D-2110 {GalNAc3K2AhxC6}ugguauGfuUfCfCfU 1349 {Phosphate}gsCfsaUfgAfaGfCfaggaAf 1350 fgcuucausgsu cAfuaccasusu D-2111 {GalNAc3K2AhxC6}guauguUfcCfUfGfC 1351 {Phosphate}gsCfsaUfgAfAfgcagGfaAf 1352 fuucausgsu cauacsusu D-2112 {GalNAc3K2AhxC6}guauguUfcCfUfGfC 1353 {Phosphate}gsCfsaUfgAfAfgcagGfaAf 1354 fuucaugcs{invAb} cauacsusu D-2113 {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCf 1355 {Phosphate}asUfsgUfaGfAfagggCfaUf 1356 CfUfucUfaCfaUfsusUf gAfaGfcsUfsu D-2114 {GalNAc3K2AhxC6}GfcUfuCfaUfgCfcC 1357 {Phosphate}asUfsgUfaGfAfagGfgCfaU 1358 fUfucUfaCfaUfsusUf fgAfaGfcsUfsu D-2115 {GalNAc3K2AhxC6}gcuucaUfgCfCfCfU 1359 {Phosphate}asusguaGfAfagggCfaUfga 1360 fucuacaususu agcsusu D-2116 {GalNAc3K2AhxC6}gcuucaUfgCfCfCfU 1361 {Phosphate}asusguaGfAfagggCfaUfga 1362 fucUfaCfaUfsusUf agcsusu D-2117 {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCf 1363 {Phosphate}asusguaGfAfagggCfaUfga 1364 CfUfucuacaususu agcsusu D-2118 {GalNAc3K2AhxC6}gcuucaUfgCfCfCfU 1365 {Phosphate}asusguaGfAfagggCfaUfgA 1366 fucuacaususu faGfcsUfsu D-2119 {GalNAc3K2AhxC6}gcuucaUfgCfCfCfU 1367 {Phosphate}asUfsgUfaGfAfagggCfaUf 1368 fucuacaususu gAfaGfcsUfsu D-2120 {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCf 1369 {Phosphate}asusguaGfAfagggCfaUfga 1370 CfUfucUfaCfaUfsusUf agcsusu D-2121 {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCf 1371 {Phosphate}asUfsgUfaGfAfagggCfaUf 1372 CfUfucuacaususu gaagcsusu D-2122 {GalNAc3K2AhxC6}gcuucaUfgCfCfCfU 1373 {Phosphate}asusguaGfAfagggCfaUfgA 1374 fucUfaCfaUfsusUf faGfcsUfsu D-2123 {GalNAc3K2AhxC6}gcuucaUfgCfCfCfU 1375 {Phosphate}asUfsgUfaGfAfagggCfaUf 1376 fucUfaCfaUfsusUf gaagcsusu D-2124 {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCf 1377 {Phosphate}asusguaGfAfagggCfaUfgA 1378 CfUfucuacaususu faGfcsUfsu D-2125 {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCf 1379 {Phosphate}asUfsgUfaGfAfagggCfaUf 1380 CfUfucuacaususu gAfaGfcsUfsu D-2126 {GalNAc3K2AhxC6}gcuucaUfgCfCfCfU 1381 {Phosphate}asUfsgUfaGfAfagggCfaUf 1382 fucUfaCfaUfsusUf gAfaGfcsUfsu D-2127 {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCf 1383 {Phosphate}asUfsgUfaGfAfagggCfaUf 1384 CfUfucUfaCfaUfsusUf gaagcsusu D-2128 {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCf 1385 {Phosphate}asusguaGfAfagggCfaUfgA 1386 CfUfucUfaCfaUfsusUf faGfcsUfsu D-2129 {GalNAc3K2AhxC6}gcuucaUfg[dC]CfC 1387 {Phosphate}asUfsgUfaGfAfagggCfaUf 1388 fuucuacaususu gaagcsusu D-2130 {GalNAc3K2AhxC6}gcuucaUfgCfCf[dC] 1389 {Phosphate}asUfsgUfaGfAfagggCfaUf 1390 Ufucuacaususu gaagcsusu 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{Phosphate}usCfsaGfgUfGfaaagAfgCf 1868 uCfUfUfUfcaccugasusu aaugususu D-2370 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1869 asUfsgUfaGfAfaaggCfaUfgaagcagsusu 1870 UfUfucuacas{invAb} D-2371 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1871 asUfsgUfaGfAfaaggCfaUfgaagcasgsus 1872 UfUfucuacas{invAb} u D-2372 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1873 asUfsgsUfaGfAfaaggCfaUfgaagcagsus 1874 UfUfucuacas{invAb} u D-2373 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1875 asUfsgUfaGfAfaaggCfaUfgaagcagusu 1876 UfUfucuacas{invAb} D-2374 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1877 asUfsgsUfaGfAfaaggCfaUfgaagcagusu 1878 UfUfucuacas{invAb} D-2375 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1879 asUfgUfaGfAfaaggCfaUfgaagcagsusu 1880 UfUfucuacas{invAb} D-2376 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1881 asUfgUfaGfAfaaggCfaUfgaagcasgsusu 1882 UfUfucuacas{invAb} D-2377 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1883 asUfsgUfaGfAfaaggCfaUfgaagcagsusu 1884 UfUfucuacsas{invAb} D-2378 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1885 asUfsgUfaGfAfaaggCfaUfgaagcagsusu 1886 UfUfucuascsas{invAb} D-2379 {sGalNAc3K2AhxC6}csugcuucaUfgCfC 1887 asUfsgUfaGfAfaaggCfaUfgaagcagsusu 1888 fUfUfucuacas{invAb} D-2380 {sGalNAc3K2AhxC6}csusgcuucaUfgCf 1889 asUfsgUfaGfAfaaggCfaUfgaagcagsusu 1890 CfUfUfucuacas{invAb} D-2381 {sGalNAc3K2AhxC6}[invAb]cuucauGf 1891 {Phosphate}asCfsuGfuAfGfaaagGfcAf 1892 cCf[dT]UfUfcuacagususu ugaagsusu D-2382 {sGalNAc3K2AhxC6}[invAb]cuucauGf 1893 {Phosphate}asCfsuGfuAfGfaaAfgGfcA 1894 cCfUfUfUfcuacagususu fugaagsusu D-2383 {sGalNAc3K2AhxC6}[invAb]cuucauGf 1895 {Phosphate}asCfsuGfuAfGfaaagGfcAf 1896 cCfuUfUfcuacagususu ugaagsusu D-2384 {sGalNAc3K2AhxC6}[invAb]cuucauGf 1897 {Phosphate}asCfsuGfuaGfaaagGfcAfu 1898 cCfUfUfUfcuacagususu gaagsusu D-2385 {sGalNAc3K2AhxC6}[invAb]cuucauGf 1899 [Phosphate}asCfsuguaGfaaagGfcAfug 1900 cCfUfUfUfcuacagususu aagsusu D-2386 {sGalNAc3K2AhxC6}[invAb]cuucauGf 1901 {Phosphate}asCfsuGfuAfGfaaagGfcAf 1902 c[dC]UfUfUfcuacagususu ugaagsusu D-2387 {sGalNAc3K2AhxC6}[invAb]cuucauGf 1903 {Phosphate}asCfsuGfuAfGfaaagGfcAf 1904 cCfUf[dT]Ufcuacagususu ugaagsusu D-2388 {sGalNAc3K2AhxC6}[invAb]gcuucaUf 1905 {Phosphate}asUfsgUfaGfAfauccCfaUf 1906 gGfGfAfUfucuacaususu gaagcsusu D-2389 {sGalNAc3K2AhxC6}[invAb]cuucauGf 1907 {Phosphate}asCfsuGfuAfGfauucGfcAf 1908 cGfAfAfUfcuacagususu ugaagsusu D-2390 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1909 {Phosphate}usUfsgUfaGfAfaaggCfaUf 1910 UfUfucuacas{invAb} gaagcagsusu D-2391 {sGalNAc3K2AhxC6}[mvAb]cugcuuca 1911 {Phosphate}usUfsgUfaGfAfaaggCfaUf 1912 UfgCfCfUfUfucuacsasa gaagcagsusu D-2392 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1913 {Phosphate}usUfsgUfaGfAfaaggCfaUf 1914 UfUfucuacas{invDA} gaagcagsusu D-2393 {sGalNAc3K2AhxC6}cugcuucaUfgGfGf 1915 {Phosphate}asUfsgUfaGfAfauccCfaUf 1916 AfUfucuacas{invAb} gaagcagsusu D-2394 {sGalNAc3K2AhxC6}[invAb]cugcuuca 1917 {Phosphate}asUfsgUfaGfAfauccCfaUf 1918 UfgGfGfAfUfucuacsasu gaagcagsusu D-2395 {sGalNAc3K2AhxC6}[invAb]CfuUfcAf 1919 {Phosphate}asCfsuGfuAfGfaaagGfcAf 1920 uGfcCfUfUfUfcuAfcAfgUfsusUf ugaagsusu D-2396 {sGalNAc3K2AhxC6}[invAb]cuucauGf 1921 {Phosphate}asCfsuGfuAfGfaaagGfcAf 1922 cCfUfUfUfcuAfcAfgUfsusUf uGfaAfgsUfsu D-2397 {sGalNAc3K2AhxC6}[invAb]gcuucaUf 1923 asUfsgUfaGfAfaaggCfaUfgaagcsusu 1924 gCfCfUfUfucuacaususu D-2398 {sGalNAc3K2AhxC6}[invAb]ugcuucau 1925 asCfsuGfuAfGfaaagGfcAfugaagcasusu 1926 GfcCfUfUfUfcuacags{invAb} D-2399 {sGalNAc3K2AhxC6}[invAb]cuucauGf 1927 {Phosphate}asCfsuGfuAfGfaaagGfcAf 1928 cCfUfUfUfcuacagususu uGfaAfgsUfsu D-2400 {sGalNAc3K2AhxC6}ugcuucauGfcCfUf 1929 asCfsuGfuAfGfaaagGfcAfugaagcasusu 1930 UfUfcuAfcAfgs{invAb} D-2401 {sGalNAc3K2AhxC6}ugcuucauGfcCfUf 1931 asCfsuGfuAfGfaaAfgGfcAfugaagcasus 1932 UfUfcuacags{invAb} u D-2402 {sGalNAc3K2AhxC6}ugcuucauGfcCfUf 1933 asCfsuguaGfaaagGfcAfugaagcasusu 1934 UfUfcuacags{invAb} D-2403 {sGalNAc3K2AhxC6}[invAb]cuucauGf 1935 asCfsuGfuAfGfaaagGfcAfugaagsusu 1936 cCfUfUfUfcuacagususu D-2404 {sGalNAc3K2AhxC6}ugcuucauGfcCfUf 1937 asCfsuGfuAfGfaaagGfcAfugaagcasusu 1938 UfUfcuacags{invAb} D-2405 {GalNAc3K2AhxC6}augccuuuCfuAfCfA 1939 {Phosphate}asGfscCfaCfUfguAfgAfaA 1940 fGfuggcusus{invAb} fgGfcAfuGfasUfsu D-2406 {sGalNAc3K2AhxC6}ucaugccuUfuCfUf 1941 asGfscCfaCfUfguagAfaAfggcaugasusu 1942 AfCfaguggcs{invAb} D-2407 {sGalNAc3K2AhxC6}[invAb]augccuUf 1943 asGfscCfaCfUfguagAf aAfggcaususu 1944 uCfUfAfCfaguggcususu D-2408 {sGalNAc3K2AhxC6}[invAb]augccuUf 1945 asGfscCfaCfUfguagAfaAfggcaususu 1946 uCfUfAfCfagUfgGfcUfsusUf D-2409 {sGalNAc3K2AhxC6}[invAb]ucaugccu 1947 asGfscCfaCfUfguagAfaAfggcaugasusu 1948 UfuCfUfAfCfaguggscsu D-2410 {sGalNAc3K2AhxC6}ucaugccuUfuCfUf 1949 asGfscCfaCfUfguagAfaAfggcaugasusu 1950 AfCfagUfgGfcs{invAb} D-2411 {sGalNAc3K2AhxC6}[invAb]augccuUf 1951 asGfscCfaCfUfguAfgAfaAfggcaususu 1952 uCfUfAfCfaguggcususu D-2412 {sGalNAc3K2AhxC6}ucaugccuUfuCfUf 1953 asGfscCfaCfUfguAfgAfaAfggcaugasus 1954 AfCfaguggcs{invAb} u D-2413 {sGalNAc3K2AhxC6}ucaugccuUfuCfUf 1955 asGfsccacUfguagAfaAfggcaugasusu 1956 AfCfaguggcs{invAb} D-2414 {sGalNAc3K2AhxC6}[invAb]ucaugcuu 1957 asGfscCfaCfUfguagAfaAfggcaugasusu 1958 UfuCfUfAfCfaguggcs{invAb} D-2415 {sGalNAc3K2AhxC6}[invAb]augccuUf 1959 usGfscCfaCfUfguagAfaAfggcaususu 1960 uCfUfAfCfaguggcasusu D-2416 {sGalNAc3K2AhxC6}ucaugccuUfuCfUf 1961 usGfscCfaCfUfguagAfaAfggcaugasusu 1962 AfCfaguggcs{invAb} D-2417 {sGalNAc3K2AhxC6}[invAb]ucaugccu 1963 usGfscCfaCfUfguagAfaAfggcaugasusu 1964 UfuCfUfAfCfaguggscsa D-2418 {sGalNAc3K2AhxC6}ucaugccuUfuCfUf 1965 usGfscCfaCfUfguagAfaAfggcaugasusu 1966 AfCfaguggcs{invDA} D-2419 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1967 asUfsguagAfaaggCfaUfgaagcagsusu 1968 UfUfucuacas{invAb} D-2420 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1969 usUfsguagAfaaggCfaUfgaagcagsusu 1970 UfUfucuacas{invAb} D-2421 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1971 usUfsguagAfaaggCfaUfgaagcagsusu 1972 UfUfucuacas{invDA} D-2422 {sGalNAc3K2AhxC6}ucaugccuUfuCfUf 1973 usGfsccacUfguagAfaAfggcaugasusu 1974 AfCfaguggcs{invAb} D-2423 {sGalNAc3K2AhxC6}ucaugccuUfuCfUf 1975 usGfsccacUfguagAfaAfggcaugasusu 1976 AfCfaguggcs{invDA} D-2424 {sGalNAc3K2AhxC6}[invAb]cuucauGf 1977 {Phosphate}asCfsuGfuAfGfaaAfgGfcA 1978 cCfuUfUfcuacagususu fugaagsusu D-2425 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1979 asUfsgUfa[Ab]AfaaggCfaUfgaagcagsu 1980 UfUfucuacas{invAb} su D-2426 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1981 asUfsgua[Ab]AfaaggCfaUfgaagcagsus 1982 UfUfucuacas{invAb} u D-2427 {sGalNAc3K2AhxC6}ugcuucauGfcCfUf 1983 asCfsuGfu[Ab]GfaaagGfcAfugaagcasu 1984 UfUfcuacags{invAb} su D-2428 {sGalNAc3K2AhxC6}ugcuucauGfcCfUf 1985 asCfsugu[Ab]GfaaagGfcAfugaagcasus 1986 UfUfcuacags{invAb} u D-2429 {sGalNAc3K2AhxC6}ucaugccuUfuCfUf 1987 asGfscCfa[Ab]UfguagAfaAfggcaugasu 1988 AfCfaguggcs{invAb} su D-2430 {sGalNAc3K2AhxC6}ucaugccuUfuCfUf 1989 asGfscca[Ab]UfguagAfaAfggcaugasus 1990 AfCfaguggcs{invAb} u D-2431 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1991 asUfs[GNA- 1992 UfUfucuacas{invAb} G]uagAfaaggCfaUfgaagcagsusu D-2432 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 1993 asUfsg[GKA- 1994 UfUfucuacas{invAb} U]agAfaaggCfaUfgaagcagsusu D-2433 {sGalNAc3K2AhxC6}cugcuucaUfgCfCf 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cugcuucaUfgCfCfUfsUfsucuacas{invAb} 2199 asUfsguagAf[sGNA- 2200 A]aggCfaUfgaagcagsusu D-2536 cugcuucaUfgCfCfUfUfsucuacas{invAb} 2201 asUfsguagAfs[sGNA- 2202 A]aggCfaUfgaagcagsusu D-2537 cugcuucaUfgCfCfUfsUfucuacas{invAb} 2203 asUfsguagAfs[sGNA- 2204 A]aggCfaUfgaagcagsusu D-2538 cugcuucaUfgCfCfUf[LNA- 2205 asUfsguagAf[GNA- 2206 T]ucuacas{invAb} A]aggCfaUfgaagcagsusu D-2539 cugcuucaUfgCfCf[LNA- 2207 asUfsguagAf[GNA- 2208 T]Ufucuacas{invAb} A]aggCfaUfgaagcagsusu D-2540 cugcuucaUfgCfCfUfUf[LNA- 2209 asUfsguagAf[GNA- 2210 T]cuacas{invAb} A]aggCfaUfgaagcagsusu D-2541 cugcuucaUfgCfCfUfUfuc[LNA- 2211 asUfsgu[GNA- 2212 T]acas{invAb} A]gAfaaggCfaUfgaagcagsusu D-2542 cugcuucaUfgCfCfUfUfucu[LNA- 2213 asUfsg[GNA- 2214 A]cas{invAb} U]agAfaaggCfaUfgaagcagsusu D-2543 cugcuucaUfgCfCfUfUfucuac[sLNA- 2215 as[sGNA- 2216 A]{invAb} U]guagAfaaggCfaUfgaagcagsusu D-2544 cugcuucaUfgCfCfUfUf[LNA- 2217 asUfsguag[Ab]aaggCfaUfgaagcagsusu 2218 T]cuacas{invAb} D-2545 cugcuucaUfgCfCfUfUfuc[LNA- 2219 asUfsgu[Ab]gAfaaggCfaUfgaagcagsus 2220 T]acas{invAb} u D-2546 cugcuucaUfgCfCfUfUfucu[LNA- 2221 asUfsg[Ab]agAfaaggCfaUfgaagcagsus 2222 A]cas{invAb} u D-2547 cugcuucaUfgCfCfUfUfucUfaCf 2223 asUfsgUfagAf[GNA- 2224 as{invAb} A]aggCfaUfgaagcagsusu D-2548 cugcuucaUfgCfCfUfUfucUfaCf 2225 asUfsguagAf[GNA- 2226 as{invAb} A]aggCfaUfgaagcagsusu D-2549 cugcuucaUfgCfCfUfs[LNA- 2227 asUfsguagAf[GNA- 2228 T]ucuacas{invAb} A]aggCfaUfgaagcagsusu D-2550 cugcuucaUfgCfCfUfs[sLNA- 2229 asUfsguagAf[GNA- 2230 T]ucuacas{invAb} A]aggCfaUfgaagcagsusu D-2551 cugcuucaUfgCfCfUf[LNA- 2231 asUfsguagAf[sGNA- 2232 T]ucuacas{invAb} A]aggCfaUfgaagcagsusu D-2552 cugcuucaUfgCfCfUf[LNA- 2233 asUfsguagAfs[GNA- 2234 T]ucuacas{invAb} A]aggCfaUfgaagcagsusu D-2553 usgscuucauGfcCfUfUfUfau[LNA- 2235 usCfsug[Ab]aGfaaagGfcAfugaagcasus 2236 A]cags{invDA} u D-2554 usgscuucauGfcCfUfUfUfc[LNA- 2237 usCfsugu[Ab]GfaaagGfcAfugaagcasus 2238 T]acags{invDA} u D-2555 usgscuucauGfcCfUfUf[LNA- 2239 usCfsuguaGf[Ab]aagGfcAfugaagcasus 2240 T]cuacags{invDA} u D-2556 usgscuucauGfcCfUfUfUfcuac[LNA- 2241 usCfs[GNA- 2242 A]gs{invDA} U]guaGfaaagGfcAfugaagcasusu D-2557 usgscuucauGfcCfUfUfUfau[LNA- 2243 usCfsug[GNA- 2244 A]cags{invDA} U]aGfaaagGfcAfugaagcasusu D-2558 usgscuucauGfcCfUfUfUfc[LNA- 2245 usCfsugu[GNA- 2246 T]acags{invDA} A]GfaaagGfcAfugaagcasusu D-2559 usgscuucauGfcCfUfUfUfcuaca[sLNA- 2247 us[Ab]uguaGfaaagGfcAfugaagcasusu 2248 G]{invDA} D-2560 usgscuucauGfcCfUfUfUfcuac[LNA- 2249 usCfs[Ab]guaGfaaagGfcAfugaagcasus 2250 A]gs{invDA} u D-2561 ucaugccuUfuCfUfAfCfa[LNA- 2251 asGfscca[Ab]UfguagAfaAfggcaugasus 2252 G]uggcs{invAb} u D-2562 ucaugccuUfuCfUfAfCfag[LNA- 2253 asGfscca[Ab]UfguagAfaAfggcaugasus 2254 T]ggcs{invAb} u D-2563 ucaugccuUfuCfUfAfCfasgsugg 2255 asGfscca[Ab]UfguagAfaAfggcaugasus 2256 cs{invAb} u D-2564 {GalNAc3K2AhxC6}GfgUfaUfgUfuCfCf 2349 {Phosphate}asAfsuGfaAfGfcaggAfaCf 2350 UfGfcuUfcAfuUfsusUf aUfaCfcsUfsu D-2565 {GalNAc3K21thxC6}GfgUfaUfgUfuCf 2351 {Phosphate}usAfsuGfaAfGfcaggAfaCf 2352 CfUfGfcuUfcAfuAfsusUf aUfaCfcsUfsu D-2566 {GalNAc3K2AhxC6}GfuAfuGfuUfcCfUf 2353 {Phosphate}asCfsaUfgAfAfgcagGfaAf 2354 GfCfuuCfaUfgUfsusUf cAfuAfcsUfsu D-2567 {GalNAc3K2AhxC6}UfaUfgUfuCfcUfGf 2355 {Phosphate}usGfscAfuGfAfagcaGfgAf 2356 CfUfucAfuGfcAfsusUf aCfaUfasUfsu D-2568 {GalNAc3K2AhxC6}AfuGfuUfcCfuGfCf 2357 {Phosphate}asGfsgCfaUfGfaagcAfgGf 2358 UfUfcaUfgCfcUfsusUf aAfcAfusUfsu D-2569 {GalNAc3K2AhxC6}UfgUfuCfcUfgCfUf 2359 {Phosphate}asAfsgGfcAfUfgaagCfaGf 2360 UfCfauGfcCfuUfsusUf gAfaCfasUfsu D-2570 {GalNAc3K2AhxC6}GfuUfcCfuGfaUfUf 2361 {Phosphate}asAfsaGfgCfAfugaaGfcAf 2362 CfAfugCfcUfuUfsusUf gGfaAfcsUfsu D-2571 {GalNAc3K2AhxC6}UfuCfcUfgCfuUfCf 2363 {Phosphate}asAfsaAfgGfCfaugaAfgCf 2364 AfUfgcCfuUfuUfsusUf aGfgAfasUfsu D-2572 {GalNAc3K2AhxC6}UfuCfcUfgCfuUfCf 2365 {Phosphate}usAfsaAfgGfCfaugaAfgCf 2366 AfUfgcCfuUfuAfsusUf aGfgAfasUfsu D-2573 {GalNAc3K2AhxC6}UfcCfuGfaUfuCfAf 2367 {Phosphate}asGfsaAfaGfGfcaugAfaGf 2368 UfGfccUfuUfcUfsusUf cAfgGfasUfsu D-2574 {GalNAc3K2AhxC6}CfcUfgCfuUfcAfUf 2369 {Phosphate}usAfsgAfaAfGfgcauGfaAf 2370 GfCfcuUfuCfuAfsusUf gCfaGfgsUfsu D-2575 {GalNAc3K2AhxC6}CfuGfcUfuCfaUfGf 2371 {Phosphate}asUfsaGfaAfAfggcaUfgAf 2372 CfCfuuUfcUfaUfsusUf aGfcAfgsUfsu D-2576 {GalNAc3K2AhxC6}CfuGfcUfuCfaUfGf 2373 {Phosphate}usUfsaGfaAfAfggcaUfgAf 2374 CfCfuuUfcUfaAfsusUf aGfcAfgsUfsu D-2577 {GalNAc3K2AhxC6}UfgCfuUfcAfuGfCf 2375 {Phosphate}usGfsuAfgAfAfaggcAfuGf 2376 CfUfuuCfuAfcAfsusUf aAfgCfasUfsu D-2578 {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCf 2377 {Phosphate}asUfsgUfaGfAfaaggCfaUf 2378 UfUfucUfaCfaUfsusUf gAfaGfcsUfsu D-2579 {GalNAc3K2AhxC6}GfcUfuCfaUfgCfCf 2379 {Phosphate}usUfsgUfaGfAfaaggCfaUf 2380 UfUfucUfaCfaAfsusUf gAfaGfcsUfsu D-2580 {GalNAc3K2AhxC6}CfuUfcAfuGfcCfUf 2381 {Phosphate}asCfsuGfuAfGfaaagGfcAf 2382 UfUfcuAfcAfgUfsusUf uGfaAfgsUfsu D-2581 {GalNAc3K2AhxC6}UfuCfaUfgCfcUfUf 2383 {Phosphate}asAfscUfgUfAfgaaaGfgCf 2384 UfCfuaCfaGfuUfsusUf aUfgAfasUfsu D-2582 {GalNAc3K2AhxC6}UfuCfaUfgCfcUfUf 2385 {Phosphate}usAfscUfgUfAfgaaaGfgCf 2386 UfCfuaCfaGfuAfsusUf aUfgAfasUfsu D-2583 {GalNAc3K2AhxC6}UfcAfuGfcCfutifUf 2387 {Phosphate}asCfsaCfuGfGfagaaAfgGf 2388 cfUfacAfgUfgUfsusUf cAfuGfasUfsu D-2584 {GalNAc3K2AhxC6}UfcAfuGfcCfuUfUf 2389 {Phosphate}usCfsaCfuGfUfagaaAfgGf 2390 CfUfacAfgUfgAfsusUf cAfuGfasUfsu D-2585 {GalNAc3K2AhxC6}CfaUfgCfcUfuUfCf 2391 {Phosphate}asCfscAfcUfGfuagaAfaGf 2392 UfAfcaGfuGfgUfsusUf gCfaUfgsUfsu D-2586 {GalNAc3K2AhxC6}CfaUfgCfcUfuUfCf 2393 {Phosphate}usCfscAfcUfGfuagaAfaGf 2394 UfAfcaGfuGfgAfsusUf gCfaUfgsUfsu D-2587 {GalNAc3K2AhxC6}AfuGfcCfuUfuCfUf 2395 {Phosphate}asGfscCfaCfUfguagAfaAf 2396 AfCfagUfgGfcUfsusUf gGfcAfusUfsu D-2588 {GalNAc3K2AhxC6}AfuGfcCfuUfuCfUf 2397 {Phosphate}usGfscCfaCfUfguagAfaAf 2398 AfCfagUfgGfcAfsusUf gGfcAfusUfsu D-2589 {GalNAc3K2AhxC6}UfgCfcUfuUfcUfAf 2399 {Phosphate}asGfsgCfcAfCfuguaGfaAf 2400 CfAfguGfgCfcUfsusUf aGfgCfasUfsu D-2590 {GalNAc3K2AhxC6}GfcCfuUfuCfuAfCf 2401 {Phosphate}asAfsgGfcCfAfcuguAfgAf 2402 AfGfugGfcCfuUfsusUf aAfgGfcsUfsu D-2591 {GalNAc3K2AhxC6}GfgUfaUfgUfuCfCf 2403 {Phosphate}gsAfsuGfaAfGfcaggAfaCf 2404 UfGfcuUfcAfuCfsusUf aUfaCfcsUfsu D-2592 {GalNAc3K2AhxC6}GfuAfuGfuUfcCfUf 2405 {Phosphate}gsGfsaUfgAfAfgcagGfaAf 2406 GfCfuuCfaUfcCfsusUf cAfuAfcsUfsu D-2593 {GalNAc3K2AhxC6}UfaUfgUfuCfcUfGf 2407 [Phosphate}gsGfsgAfuGfAfagcaGfgAf 2408 CfUfucAfuCfcCfsusUf aCfaUfasUfsu D-2594 {GalNAc3K2AhxC6}AfuGfuUfcCfuGfCf 2409 {Phosphate}gsGfsgGfaUfGfaagcAfgGf 2410 UfUfcaUfcCfcCfsusUf aAfcAfusUfsu D-2595 {GalNAc3K2AhxC6}UfgUfuCfcUfgCfUf 2411 {Phosphate}asGfsgGfgAfUfgaagCfaGf 2412 UfCfauCfcCfcUfsusUf gAfaCfasUfsu D-2596 {GalNAc3K2AhxC6}GfuUfcCfuGfcUfUf 2413 {Phosphate}asAfsgGfgGfAfugaaGfcAf 2414 CfAfucCfcCfuUfsusUf gGfaAfcsUfsu D-2597 {GalNAc3K2AhxC6}UfuCfcUfgCfuUfCf 2415 {Phosphate}gsAfsaGfgGfGfaugaAfgCf 2416 AfUfccCfcUfuCfsusUf aGfgAfasUfsu D-2598 {GalNAc3K2AhxC6}UfcCfuGfcUfuCfAf 2417 {Phosphate}asGfsaAfgGfGfgaugAfaGf 2418 ufCfccCfuUfcUfsusUf cAfgGfasUfsu D-2599 {GalNAc3K2AhxC6}CfcUfgCfuUfcAfUf 2419 {Phosphate}usAfsgAfaGfGfggauGfaAf 2420 cfCfccUfuefuAfsusUf gCfaGfgsUfsu D-2600 {GalNAc3K2AhxC6}CfuGfcUfuCfaUfCf 2421 {Phosphate}gsUfsaGfaAfGfgggaUfgAf 2422 CfCfcuUfcUfaCfsusUf aGfcAfgsUfsu D-2601 {GalNAc3K2AhxC6}UfgCfuUfcAfuCfCf 2423 {Phosphate}usGfsuAfgAfAfggggAfuGf 2424 CfCfuuCfuAfcAfsusUf aAfgCfasUfsu D-2602 {GalNAc3K2AhxC6}GfcUfuCfaUfcCfCf 2425 {Phosphate}csUfsgUfaGfAfagggGfaUf 2426 CfUfucUfaCfaGfsusUf gAfaGfcsUfsu D-2603 {GalNAc3K2AhxC6}CfuUfcAfuCfcCfCf 2427 {Phosphate}asCfsuGfuAfGfaaggGfgAf 2428 UfUfcuAfcAfgUfsusUf uGfaAfgsUfsu D-2604 {GalNAc3K2AhxC6}UfuCfaUfcCfcCfUf 2429 {Phosphate}csAfscUfgUfAfgaagGfgGf 2430 UfCfuaCfaGfuGfsusUf aUfgAfasUfsu D-2605 {GalNAc3K2AhxC6}UfcAfuCfcCfcUfUf 2431 {Phosphate}csCfsaCfuGfUfagaaGfgGf 2432 CfUfacAfgUfgGfsusUf gAfuGfasUfsu D-2606 {GalNAc3K2AhxC6}CfaUfcCfcCfuUfCf 2433 {Phosphate}gsCfscAfcUfGfuagaAfgGf 2434 UfAfcaGfuGfgCfsusUf gGfaUfgsUfsu D-2607 {GalNAc3K2AhxC6}AfuCfcCfcUfuCfUf 2435 {Phosphate}gsGfscCfaCfUfguagAfaGf 2436 AfCfagUfgGfcCfsusUf gGfgAfusUfsu D-2608 {GalNAc3K2AhxC6}UfcCfcCfuUfcUfAf 2437 {Phosphate}asGfsgCfcAfCfuguaGfaAf 2438 CfAfguGfgCfcUfsusUf gGfgGfasUfsu D-2609 {GalNAc3K2AhxC6}CfcCfcUfuCfuAfCf 2439 {Phosphate}asAfsgGfcCfAfcuguAfgAf 2440 AfGfugGfcCfuUfsusUf aGfgGfgsUfsu

Example 2: Efficacy of Select PNPLA3 siRNA Molecules in RNA FISH Assay

A panel of fully chemically modified siRNA, including siRNA spanning the rs738409 and/or the rs738408 SNP in PNPLA3, were prepared and tested for potency and selectivity of mRNA knockdown in vitro. Each siRNA duplex consisted of two strands, the sense or ‘passenger’ strand and the antisense or ‘guide’ strand. The strands are 21 or 23 nucleotides in length with 19 complementary base pairs. In some instances, there are two base pair 3′ overhangs. The siRNA were prepared with substitution of the natural 2′-OH in the ribose of each nucleotide with either a 2′-OMe or 2′—F group. Optionally, phosphosdiester internucleotide linkages at one or both strands were replaced with phosphorothioates to reduce degradation by exonucleases.

The efficacy of each of the siRNA molecules in reducing PNPLA3 expression was assessed using a 384-well format in vitro siRNA transfection assay followed by a fluorescent in situ hybridization targeting ribonucleic acid molecules (RNA FISH) assay to determine IC50 and maximum activity values. This assay was performed on human hepatocellular carcinoma cell line Hep3B cells (ATCC HB-8064) and Chinese hamster ovary (CHO) cells expressing human PNPLA3 I1481. Human hepatocellular carcinoma HepB3 was maintained in EMEM media (ATCC 30-2003) supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic at 37° C. and 5% CO2. CHO cells expressing human PNPLA3 I1481 was maintained in media containing 50% CD-CHO (Life Technologies), 50% Ex-Cell CHO 5 Medium (Sigma), 8 mM L-Glutamine, 1×HT, 1% antibiotic/antimycotic, and 10 μg/mL Puromycin at 37° C. and 5% CO2.

For Hep3B cell assays, transfection complexes of the siRNA molecules and the Lipofectamine RNAiMAX transfection reagent (Life Technologies) in EMEM media (ATCC 30-2003) were prepared in 384-well plates (PerkinElmer), at 10 μg per well, in accordance with manufacturer's recommendations. For CHO human cell assays, transfection complexes of the siRNA molecules and the Lipofectamine RNAiMAX transfection reagent in F12K media (Mediatech) were prepared in 384-well plates, at 10 μg per well, in accordance with manufacturer's recommendations. Cells were diluted to 67,000 cells/ml in antibiotic/antimycotic-free media and 30 μl added to each well, a final density of 2000 cells/well in 40 μl media. After a 20 minute incubation at room temperature, plates were transferred to a 37° C. and 5% CO2 incubator. Hep3B cell transfection assays were incubated for 72 hours and CHO human PNPLA3 I1481 transfection assays were incubated for 48 hours.

At harvest, the cells were fixed in an 8% formaldehyde fixative solution (Thermo Scientific) for 15 minutes at room temperature. The plates were then subjected to dehydration with sequential 50%, 70%, and 100% ethanol washes. Plates were then sealed and stored at −20° C.

The RNA FISH assay was performed using the Affymetrix QuantiGene® View RNA HC Screening Assay kit (QVP0011), the Affymetrix View HC Signal Amplification Kit 3-plex (QVP0213), and Affymetrix gene specific probes: PNPLA3 Human 0.33 mL View RNA Type 6 (650 label) VA6-20279-01 and PPM Human 0.44 mL View RNA Type 1 (488 label) VA1-10148-01.

Plates were first rehydrated with sequential 100%, 70%, and 50% ethanol washes. Cells were then washed with PBS, and then permeabilized and protease-digested according to the kit instructions. The target Working Probe Sets were prepared according to the manufacturer's protocol, added to the wells, and incubated for 3 hours at 40° C. The manufacturer's protocol was followed for the sequential hybridizations with the Working Probe Sets, the Working PreAmps, the Working Amps, and the Working LPs. Last, nuclei counterstains were applied (Hoechst 33342 and Cell Mask Blue; Molecular Probes). Plates were incubated for 30 minutes at room temperature, washed with PBS, overlaid with 80 μl of PBS, and then the plates were sealed for imaging.

All plates were imaged on an Opera Phenix High Content Screening System (PerkinElmer), using the UV Channel for Hoechst 33342 and Cell Mask Blue, the 488 Channel for Typel probes, and the 647 Channel for Type6 probes.

RNA FISH data was analyzed using Columbus software and images were generated using Genedata Screener. The results of the assay for CHO transfected PNPLA3 I1481 are shown in Table 3. The results of the assay for CHO transfected PNPLA3 I148M are shown in Table 4. PNPLA3 knockdown provides a percentage of knockdown compared to control. Negative values indicate a decrease in PNPLA3 levels.

TABLE 3 RNA FISH assay on CHO transfected PNPLA3 I148I Duplex No. IC50 (μM) PNPLA3 knockdown (%) D-2001 .0589 −33.9 D-2002 .0158 −67.2 D-2003 .0427 −43.4 D-2004 .00835 −63.5 D-2006 .0177 −77.8 D-2008 .125 −10.7 D-2009 .00769 −45.5 D-2010 .00558 −80 D-2011 .035 −3 D-2012 >0.5 −3.7 D-2013 .036 −6.4 D-2014 .0122 −58.2 D-2015 >0.5 8 D-2016 >0.5 8 D-2017 .0153 −73.2 D-2018 .00386 −31.5 D-2019 .125 D-2020 >0.5 D-2021 >0.5 8 D-2022 >0.167 −6.8 D-2023 .0257 −36.9 D-2024 >0.5 4 D-2025 >0.5 −2.5 D-2026 .022 −35.1 D-2027 .00172 −16.5 D-2028 >0.5 10 D-2029 .0106 −56.2 D-2032 .00205 −52.9 D-2033 .0107 −61.7 D-2034 >0.5 6 D-2035 >0.5 −3.8 D-2036 .00665 −55.9 D-2037 >0.5 4 D-2038 >0.5 10 D-2039 .0116 −23.9 D-2040 >0.5 −25.4 D-2041 >0.5 9 D-2042 .00959 −25.5 D-2043 >0.5 9 D-2044 .00552 −29 D-2045 >0.5 9 D-2046 >0.5 9 D-2047 >0.5 −5.9 D-2048 .00618 −56.3 D-2049 >0.5 12 D-2050 >0.5 10 D-2051 >0.5 −17.2 D-2052 >0.5 −8.3 D-2053 >0.5 11 D-2054 >0.5 −14.9 D-2055 >0.5 −10.6 D-2056 >0.5 10 D-2057 .00485 −59.2 D-2058 .014 −53 D-2059 >0.5 −4.9 D-2060 >0.5 18 D-2061 .00795 −44.8 D-2062 .000668 −74.6 D-2063 >0.5 -21.8 D-2064 >0.5 9 D-2065 >0.5 −10.5 D-2066 .0412 −42.2 D-2067 >0.5 10

TABLE 4 RNA FISH assay on CHO transfected PNPLA3 I148M Duplex No. IC50 (μM) PNPLA3 knockdown (%) D-2000 .0316 −29.2 D-2001 .0131 −81.8 D-2002 .00216 −90.5 D-2003 .022 −50.4 D-2004 .00429 −88 D-2005 >0.5 15 D-2006 .00301 −89.2 D-2007 >0.5 6 D-2009 .00274 −86.9 D-2010 .00203 −93.3 D-2011 .000694 −11.9 D-2012 >0.5 −18 D-2013 .011 −66.4 D-2014 .0057 −84.3 D-2015 >0.5 −13.5 D-2016 >0.5 −12.4 D-2017 .00448 −89.4 D-2018 .0104 −36.2 D-2019 .0302 −7.7 D-2020 .01 −78.9 D-2021 .00435 −83.5 D-2022 .00628 −88.6 D-2023 .0143 −44.3 D-2024 >0.5 11 D-2025 .00355 −58.2 D-2026 .000867 −39.4 D-2027 >0.5 32 D-2028 .00205 −89.9 D-2029 .0019 −94 D-2030 >0.5 −9.4 D-2031 >0.5 4

RNA FISH was also run on a hepatic cell line containing the double mutant PNPLA3-rs738408-rs738409, as well as a control wild-type cell line, Hep3B. Hep3B and HepG2 cells (purchased from ATCC) were cultured in minimal essential medium (MEM from Corning for Hep3B and EMEM from ATCC for HepG2) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (P—S, Corning). The siRNA transfection was performed as follows: 1 μL of test siRNAs and 4 μL of plain MEM or EMEM, depending on the cell line, were added to PDL-coated CellCarrier-384 Ultra assay plates (PerkinElmer) by BioMek FX (Beckman Coulter). 5 μL of Lipofectamine RNAiMAX (Thermo Fisher Scientific), pre-diluted in plain MEM or EMEM (specifically 0.035 μL RNAiMAX in 5 μL MEM for Hep3B and 0.06 μL of RNAiMAX in 5 μL EMEM for HepG2), was then dispensed into the assay plates by Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific). After 20 mins incubation of the siRNA/RNAiMAX mixture at room temperature (RT), 30 μL of either Hep3B or HepG2 cells (2000 cells per well) in MEM or EMEM supplemented with 10% FBS and 1% P—S were added to the transfection complex using Multidrop Combi Reagent Dispenser. The assay plates were incubated for 20 mins at RT prior to being placed in an incubator. Cells were then incubated for 72 hrs at 37° C. and 5% CO2. ViewRNA ISH Cell Assay was performed following manufacture's protocol (Thermo Fisher Scientific) using an in-house assembled automated FISH assay platform for liquid handling. In brief, cells were fixed in 4% formaldehyde (Thermo Fisher Scientific) for 15 mins at RT, permeabilized with detergent for 3 mins at RT and then treated with protease solution for 10 mins at RT. Incubation of target-specific probe pairs (Thermo Fisher Scientific) was done for 3 hrs, while for Preamplifiers, Amplifiers and Label Probes (Thermo Fisher Scientific) were for 1 hr each. All hybridization steps were carried out at 40° C. in Cytomat 2 C-LIN automated incubator (Thermo Fisher Scientific). After hybridization reactions, cells were stained for 30 mins with Hoechst and CellMask Blue (Thermo Fisher Scientific) and then imaged on Opera Phenix (PerkinElmer). The images were analyzed using Columbus Image Data Storage and Analysis System (PerkinElmer) to obtain mean spot counts per cell. The spot counts were normalized using the high (containing phosphate-buffered saline, Corning) and low (without target probe pairs) control wells. The normalized values against the total siRNA concentrations were plotted and the data were fit to a four-parameter sigmoidal model in Genedata Screener (Genedata) to obtain IC50 and maximum activity. The results of the assay for HepG2 cells is shown in Table 5 and the results of the assay for Hep3B cells is shown in Table 6. PNPLA3 knockdown provides a percentage of knockdown compared to control. Negative values indicate a decrease in PNPLA3 levels. In instances where a duplex was run more than once, the average IC50 is shown, with a standard deviation.

TABLE 5 RNA FISH assay on hepatic HepG2 cells Duplex No. IC50 (μM) PNPLA3 knockdown (%) D-2001 .0132 −70.5 D-2003 .0458 −46.9 D-2004 .0049 −74.2 D-2006 .00283 −69.6 D-2009 .00448 −62.2 D-2010 .00206 −52 D-2013 .00319 −71.7 D-2014 .00164 −67.4 D-2017 .00222 −60.9 D-2018 .00717 −52.7 D-2020 .00664 −68.3 D-2021 .00559 −61.5 D-2022 .004 −30.5 D-2023 >0.5 −18 D-2025 .0041 −61.9 D-2026 .00937 −34.6 D-2564 0.009 −64.021 D-2565 0.00609 −70.965 D-2566 0.00255 −52.65 D-2567 0.00584 −56.603 D-2568 0.0157 −63.002 D-2569 0.00327 −64.898 D-2570 0.00144 −55.424 D-2571 >0.1 −18.661 D-2572 0.00557 −25.668 D-2573 0.0115 −55.329 D-2574 0.00289 −69.84 D-2575 0.00378 −69.491 D-2576 0.00527 −64.841 D-2577 0.00511 −46.449 D-2578 0.0026 −67.821 D-2579 0.00402 −67.057 D-2580 0.00119 −64.422 D-2581 0.00915 −73.008 D-2582 0.000823 −77.053 D-2583 0.00851 −66.555 D-2584 0.00513 −54.442 D-2585 0.0154 −67.707 D-2586 0.00701 −69.624 D-2587 0.00732 −66.627 D-2588 0.00226 −70.854 D-2589 0.00837 −31.221 D-2590 0.0249 −41.857 D-2591 0.009 −64.021 D-2592 0.00609 −70.965 D-2593 0.00255 −52.65 D-2594 0.00584 −56.603 D-2595 0.0157 −63.002 D-2596 0.00327 −64.898 D-2597 0.00144 −55.424 D-2598 >0.1 −18.661 D-2599 0.00557 −25.668 D-2600 0.0115 −55.329 D-2601 0.00289 −69.84 D-2602 0.00378 −69.491 D-2603 0.00527 −64.841 D-2604 0.00511 −46.449 D-2605 0.0026 −67.821 D-2606 0.00402 −67.057 D-2607 0.00119 −64.422 D-2608 0.00915 −73.008 D-2609 0.000823 −77.053 D-2591 0.00851 −66.555 D-2592 0.00513 −54.442 D-2593 0.0154 −67.707 D-2594 0.00701 −69.624 D-2595 0.00732 −66.627 D-2596 0.00226 −70.854 D-2597 0.00837 −31.221 D-2598 0.0249 −41.857 D-2426 85.098 −14.902 D-2444 31.575 +/− 6.79 −68.425 +/− 6.79 D-2454 53.215 −46.785 D-2473 29.35 −70.65

TABLE 6 RNA FISH assay on hepatic Hep3B cells Duplex No. IC50 (μM) PNPLA3 knockdown (%) D-2001 .00842 −37 D-2003 .0158 −32.1 D-2004 .00266 −32.4 D-2006 .00948 −54.1 D-2009 .00228 −29.5 D-2010 .00219 −37.2 D-2013 .00524 −31.5 D-2014 .00148 −37.6 D-2017 .00333 −37.6 D-2018 .00315 −21.3 D-2020 >0.5 6 D-2021 >0.5 −1.6 D-2022 .00272 −30.9 D-2023 >0.5 24 D-2025 .0101 −30.3 D-2026 .00551 −23 D-2564 0.01 −63.199 D-2565 0.00938 −58.392 D-2566 0.00343 −61.484 D-2567 0.0175 −53.489 D-2568 >0.1 −18.367 D-2569 0.0195 −62.568 D-2570 0.0127 −77.141 D-2571 >0.1 −15.922 D-2572 >0.1 −12.434 D-2573 >0.1 −14.649 D-2574 0.0215 −52.515 D-2575 0.0203 −53.175 D-2576 0.018 −48.137 D-2577 >0.1 −16.105 D-2578 >0.1 −21.309 D-2579 >0.1 −17.510 D-2580 >0.1 −24.616 D-2581 >0.1 −13.987 D-2582 0.0574 −30.543 D-2583 >0.1 −23.990 D-2584 >0.1 −6.715 D-2585 >0.1 −17.518 D-2586 >0.1 −24.518 D-2587 0.0391 −58.478 D-2588 0.0218 −56.609 D-2589 >0.1 −17.418 D-2590 >0.1 −21.161 D-2591 0.0167 −59.366 D-2592 0.0104 −61.548 D-2593 Undefined −61.879 D-2594 0.0211 −43.856 D-2595 0.0272 −63.020 D-2596 >0.1 −10.278 D-2597 0.0546 −31.743 D-2598 Undefined −47.517 D-2599 0.00489 −70.825 D-2600 >0.1 −8.522 D-2601 0.0364 −31.836 D-2602 0.00577 −65.062 D-2603 0.01 −58.287 D-2604 0.00353 −40.649 D-2605 0.0113 −50.691 D-2606 >0.1 −5.097 D-2607 0.0261 −49.898 D-2608 >0.1 −23.747 D-2609 >0.1 −23.804 D-2426 >0.1 −15.207 D-2454 0.00187 −51.735 D-2473 >0.1 −21.333

Example 3: Droplet Digital PCR Assay of siRNA for PNPLA3-Rs738409 and PNPLA3-rs738409-rs738408

Following the manufacturers protocol, thawed human primary hepatocyte cells (Xenotech/Sekisui donor lot #HC3-38) in OptiThaw media (Xenotech cat #K8000), cells were centrifuged and post media aspiration, resuspended in OptiPlate hepatocyte media (Xenotech cat #K8200) and plated into 96 well collagen coated plates (Greiner cat #655950). Following a 2-4 hour incubation period, media was removed and replaced with OptiCulture hepatocyte media (Xenotech cat #K8300). 2-4 hours post addition of OptiCulture media, delivered GalNAc conjugated siRNAs to cells via free uptake (no transfection reagent). Cells were incubated 24-72 hours at 37° C. and 5% CO2. Cells were then lysed with Qiagen RLT buffer (79216)+1% 2-mercaptoethanol (Sigma, M-3148), and lysates were stored at −20° C. RNA was purified using a Qiagen QIACube HT instrument (9001793) and a Qiagen RNeasy 96 QIACube HT Kit (74171) according to manufacturer's instructions. Samples were analyzed using a QIAxpert system (9002340). cDNA was synthesized from RNA samples using the Applied Biosystems High Capacity cDNA Reverse Transcription kit (4368813), reactions were assembled according to manufacturer's instructions, input RNA concentration varied by sample. Reverse transcription was carried out on a BioRad tetrad thermal cycler (model #PTC-0240G) under the following conditions: 25° C. 10 minutes, 37° C. 120 minutes, 85° C. 5 minutes followed by (an optional) 4° C. infinite hold.

Droplet digital PCR (ddPCR) was performed using BioRad's QX200 AutoDG droplet digital PCR system according to manufacturer's instructions. Reactions were assembled into an Eppendorf clear 96 well PCR plate (951020303) using BioRad ddPCR Supermix for Probes (1863010), and fluorescently labeled qPCR assays for PNPLA3 (IDT Hs.PT.58.21464637, primer to probe ratio 3.6:1 and TBP (IDT Hs.PT.53a.20105486, primer to probe ratio 3.6:1) and RNase free water (Ambion, AM9937). Final primer/probe concentration is 900 nM/250 nM respectively, input cDNA concentration varied among wells. Droplets were formed using a BioRad Auto DG droplet generator (1864101) set up with manufacturer recommended consumables (BioRad DG32 cartridges 1864108, BioRad tips 1864121, Eppendorf blue 96 well PCR plate 951020362, BioRad droplet generation oil for probes 1864110 and a BioRad droplet plate assembly). Droplets were amplified on a BioRad C1000 touch thermal cycler (1851197) using the following conditions: enzyme activation 95° C. 10 minutes, denaturation 94° C. 30 seconds followed by annealing/extension 60° C. for one minute, 40 cycles using a 2° C./second ramp rate, enzyme deactivation 98° C. 10 minutes followed by (an optional) 4° C. infinite hold. Samples were then read on a BioRad QX200 Droplet Reader measuring FAM/HEX signal that correlates to PNPLA3 or TBP concentration. Data was analyzed using BioRad's QuantaSoft software package. Samples are gated by channel (fluorescent label) to determine the concentration per sample. Each sample is then expressed as the ratio of the concentration of the gene of interest (PNPLA3)/concentration of the housekeeping gene (TBP) to control for differences in sample loading. Data is then imported into Genedata Screener, where each test siRNA is normalized to the median of the neutral control wells (buffer only). IC50 values are reported in Table 7.

TABLE 7 ddPCR assay on primary hepatocyte cells Duplex No. IC50 (μM) % PNPLA3 knockdown D-2068 .0339 −49.628 D-2069 .00408 −52.997 D-2070 .00433 −42.193 D-2072 .00884 −53.16 D-2073 >2.0 −7.435 D-2078 .0044 −43.123 D-2084 0.00499 −38.791 D-2085 0.00539 −64.312 D-2086 >2.0 −14.938 D-2087 >2.0 −25.465 D-2088 0.207 −34.944 D-2089 0.0107 −38.791 D-2090 0.0218 −38.977 D-2091 0.0508 −41.209 D-2092 0.00192 −44 D-2093 0.00634 −30.233 D-2094 >2.0 4.93 D-2095 0.00181 −59.814 D-2096 0.0181 −52.807 D-2099 0.00549 −39.296 D-2100 0.0142 −55.281 D-2158 0.0681 −48.649 D-2159 0.0325 −36.036 D-2160 >0.667 −13.514 D-2161 >2.0 −24.229 D-2162 0.0726 −28.634 D-2163 >0.667 −16.3 D-2164 >2.0 −15.418 D-2165 0.00644 −26.872 D-2166 0.00192 −30.045 D-2167 >0.667 −6.726 D-2168 >2.0 −15.418 D-2169 >2.0 −13.004 D-2170 >2.0 −9.417 D-2171 0.00505 −44.395 D-2172 0.003 −55.336 D-2173 0.00598 −46.188 D-2174 >2.0 −9.009 D-2175 0.017 −27.928 D-2176 0.00452 −35.426 D-2177 >2.0 4.5 D-2178 >2.0 1.8 D-2179 >2.0 −6.306 D-2180 0.00546 −40.969 D-2181 0.00152 −43.119 D-2182 0.00317 −54.128 D-2183 0.00948 −58.079 D-2184 0.0109 −50.459 D-2185 >2.0 −7.339 D-2186 0.0021 −48.624 D-2187 >2.0 −11.009 D-2188 >2.0 1.32 D-2191 0.0984 −66.923 D-2192 0.124 −64.231 D-2193 0.138 −60.606 D-2194 0.0478 −54.182 D-2195 0.0801 −47.515 D-2199 0.0517 −62.973 D-2201 0.0517 −62.973 D-2202 0.0165 −72.404 D-2203 0.00946 −49.459 D-2204 0.0241 −58.545 D-2205 0.0382 −45.576 D-2206 0.0222 −50.946 D-2209 0.0867 −46.622 D-2212 0.358 −60 D-2216 0.0826 −58.942 D-2218 0.0242 −63.462 D-2220 0.0113 −69.333 D-2221 0.138 −55.541 D-2224 0.0198 −66.486 D-2225 0.245 −68.077 D-2228 0.155 −44.606 D-2229 0.0651 −38.909 D-2231 0.0512 −56.892 D-2232 0.0678 −67.981 D-2233 0.00802 −57.182 D-2234 0.00473 −55.947 D-2235 0.00816 −62.115 D-2236 0.00245 −51.542 D-2237 0.00495 −60 D-2238 0.00561 −63.017 D-2239 0.00453 −55.537 D-2240 0.00584 −56.116 D-2241 0.00755 −54.76 D-2242 0.0137 −56.332 D-2243 0.00329 −57.118 D-2244 0.0127 −56.909 D-2245 0.00697 −58.364 D-2246 0.00713 −56.828 D-2247 0.00875 −57.797 D-2248 0.0098 −58.59 D-2249 0.00603 −57.759 D-2250 0.0105 −62.155 D-2251 0.00521 −59.914 D-2252 0.00988 −58.678 D-2253 0.00481 −57.118 D-2254 0.00721 −56.332 D-2255 0.00788 −52.838 D-2256 0.00831 −55.455 D-2257 0.00503 −54.545 D-2258 0.00626 −54.545 D-2259 0.00401 −55.947 D-2260 0.00379 −52.423 D-2261 0.00151 −54.31 D-2262 0.00292 −53.448 D-2263 0.00607 −59.483 D-2264 0.00703 −59.504 D-2265 >4.0 −20.524 D-2266 0.0129 −32.727 D-2267 0.107 −27.273 D-2275 0.00359 −45.701 D-2276 0.00416 −43.891 D-2277 0.00218 −49.14 D-2278 0.00743 −42.986 D-2279 0.0116 −53.846 D-2280 0.00347 −36.652 D-2281 0.00449 −43.891 D-2282 0.0134 −37.557 D-2283 0.00864 −34.842 D-2284 0.00738 −49.558 D-2285 0.0202 −38.053 D-2286 0.00543 −45.487 D-2287 0.00934 −47.611 D-2288 0.00652 −55.575 D-2289 0.0259 −61.593 D-2290 0.00549 −53.805 D-2291 0.00476 −51.062 D-2292 0.0105 −42.584 D-2293 0.0059 −45.455 D-2294 0.0117 −45.646 D-2295 0.0109 −52.823 D-2296 0.01246 +/− 0.015 −59.847 +/− 15.2 D-2297 0.00828 −44.444 D-2298 0.0279 −41.346 D-2299 0.00529 −55.926 D-2300 0.0195 −50.423 D-2301 0.00838 −35.577 D-2302 0.00832 −40.865 D-2303 0.00371 −40.096 D-2304 0.00563 −38.365 D-2305 0.00639 −40.385 D-2306 0.00669 −41.25 D-2307 0.00212 −38.462 D-2308 0.00573 −37.736 D-2309 0.0645 −33.962 D-2310 0.00232 −33.019 D-2311 0.00181 −31.132 D-2312 0.0447 −45.283 D-2313 0.00655 −42.453 D-2314 0.00613 −41.509 D-2315 0.00941 −50 D-2316 0.0218 −41.784 D-2317 0.0142 −42.723 D-2318 0.0182 −31.455 D-2319 >4.0 −23.005 D-2320 0.0228 −37.089 D-2321 0.00809 −45.352 D-2322 0.0165 −44.601 D-2323 0.0184 −38.373 D-2324 0.00766 −41.627 D-2325 0.00815 −46.507 D-2326 0.0168 −48.325 D-2327 0.00663 −62.254 D-2328 0.00716 −39.367 D-2329 0.044 −52.036 D-2330 0.00282 −65.701 D-2331 0.00411 −53.991 D-2332 0.0127 −51.222 D-2333 0.012 −48.357 D-2334 0.019 −42.593 D-2335 0.00448 −47.418 D-2336 0.00944 −37.327 D-2337 0.00514 −37.327 D-2338 0.154 −38.249 D-2339 0.0089 −40.092 D-2340 0.0169 −48.148 D-2341 0.00274 −46.296 D-2342 0.0225 −42.723 D-2343 0.00222 +/− 0.00136 −43.218 +/− 4.81 D-2344 0.0136 −56.561 D-2345 0.0222 −50.226 D-2346 0.0273 −55.385 D-2347 0.0164 −41.784 D-2348 0.0314 −60.282 D-2349 0.0103 −65.1 D-2350 0.0427 −63.9

Example 4: Efficacy Screening of Select PNPLA3 siRNA Molecules in a Humanized Mouse Model

Associated adenovirus (AAV; serotype AAV8 or AAV7; endotoxin-free, prepared internally by Amgen) diluted in phosphate buffered saline (Thermo Fisher Scientific, 14190-136) to 4e11 up to 1e12 viral particles per animal, was injected intravenously into the tail vein of C57BL/6NCrl male mice (Charles River Laboratories Inc.) to drive expression of either human PNPLA3^(WT) (PNPLA3-WT), PNPLA3^(rs738409) (PNPLA3-I148M), or PNPLA3^(rs738409-rs738408) (PNPLA3-I148M DM) in the liver. Mice were generally 10-12 weeks of age and an n=4-6 animals were included per group. Every round of screening included at least two vehicle-treated control groups: AAV-empty vector and AAV-PNPLA3^(WT) or PNPLA3^(rs738409) and PNPLA3^(rs738409-rs738408) treated with vehicle. All siRNAs were tested against AAV-PNPLA3^(WT), PNPLA3^(rs738409), and/or PNPLA3^(rs738409-rs738408). Two weeks after AAV injection, mice were treated with a single dose of siRNA D-2324 (0.5 mM), via subcutaneous injection, at 0.5, 1.0, 3.0 or 5.0 milligrams per kilogram of animal, diluted in phosphate buffered saline (Thermo Fisher Scientific, 14190-136). At 8, 15, 22, 28 or 42 days post-siRNA injection, livers were collected from the animals, snap frozen in liquid nitrogen, processed for purified RNA using a QIACube HT instrument (Qiagen, 9001793) and RNeasy 96 QIACube HT kits (Qiagen, 74171) according to manufacturer's instructions. Samples were analyzed using a QIAxpert system (Qiagen, 9002340). RNA was treated with RQ1 RNase-Free DNase (Promega, M6101) and prepared for Real-Time qPCR using the TaqMan™ RNA-to-CT™ 1-Step kit (Applied Biosystems, 4392653). Real-Time qPCR was run on a QuantStudio Real-Time PCR machine. Results are based on gene expression of human PNPLA3 as normalized to mouse Gapdh (TaqMan™ assays from Invitrogen, hs00228747_m1 and 4352932E, respectively), and presented as the relative knockdown of human PNPLA3 mRNA expression compared to vehicle-treated control animals. Endogenous mouse Pnpla3 expression was determined for comparison (Invitrogen, Mm00504420_m1).

For hepatic triglyceride content analysis, approximately 0.05-0.1 milligrams of frozen liver from an animal was homogenized in one milliliter of isopropanol. After one hour of incubation on ice, samples were spun at 10,000 rpm in a microfuge, and supernatants transferred to a clean deep-well 96-well plate. Triglyceride content was determined using the colorimetric Infinity Triglyceride Reagent (Thermo Fisher Scientific, TR22421) and Triglyceride Standard (Pointe Scientific, T7531-STD) according to manufacturer's instructions. Results are presented as milligrams of triglyceride per milligrams of tissue.

All animal experiments described herein were approved by the Institutional Animal Care and Use Committee (IACUC) of Amgen and cared for in accordance to the Guide for the Care and Use of Laboratory Animals, 8^(th) Edition (National Research Council (U.S.). Committee for the Update of the Guide for the Care and Use of Laboratory Animals., Institute for Laboratory Animal Research (U.S.), and National Academies Press (U.S.) (2011) Guide for the care and use of laboratory animals. 8th Ed., National Academies Press, Washington, D.C. Mice were single-housed in an air-conditioned room at 22±2° C. with a twelve-hour light; twelve-hour darkness cycle (0600-1800 hours). Animals had ad libitum access to a regular chow diet (Envigo, 2920X) and to water (reverse osmosis-purified) via automatic watering system, unless otherwise indicated. At termination, blood was collected by cardiac puncture under deep anesthesia, and then, following Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines, euthanized by a secondary physical method. FIG. 1A-D. An example of five siRNA molecules screened for both dose-dependent mRNA knockdown and functional durability in vivo. Mice expressing human PNPLA3^(rs738409-rs738408) were treated with siRNA two-weeks after intravenous AAV injections. N=6 mice per group; data represented as the average and the standard error of the mean. (A) siRNAs were injected at 0.5, 1.0, 3.0, or 5.0 milligrams per kilogram of body weight subcutaneously into the abdomen of the mouse. Four weeks after siRNA treatment, mice were sacrificed, and the livers were collected and processed for gene expression analysis. The data represents the average relative knockdown of human PNPLA3^(rs738409-rs738408) in each group set to the vehicle-treated control group. (B) Livers from the same four-week treatment group were also processed for triglyceride content to access functional efficacy. The data represents the average milligrams of triglyceride per gram of tissue processed. (C) siRNAs were injected at 1.0 and 3.0 milligrams per kilogram of body weight subcutaneously into the abdomen of a parallel cohort of animals. Mice were harvested six weeks after siRNA treatment to compare durability of the siRNA molecules in vivo. The livers were collected and processed for gene expression analysis. The data represents the average relative knockdown of human PNPLA3^(rs738409-rs738408) in each group set to the vehicle-treated control group. (D) Livers from the same six-week treatment group were also processed for triglyceride content to access functional efficacy over time. The data represents the average milligrams of triglyceride per gram of tissue processed.

Data for relative knockdown is shown in Tables 8-12, showing relative knockdown at day 8, 15, 22, 28, and 42, respectively and the various doses. PNPLA3 knockdown is a percentage, with negative values indicating a decrease in PNPLA3 levels.

TABLE 8 Day 8 PNPLA3 knockdown assay AAV Dose PNPLA3 Duplex particles/ administered knockdown number AAV vector animal (mg/kg) (%) D-2092 PNPLA3-I148M 1.00E+12 3 −91.51 D-2092 PNPLA3-I148M 1.00E+12 5 −92.64 D-2095 PNPLA3-I148M 1.00E+12 3 −79.21 D-2095 PNPLA3-I148M 1.00E+12 5 −86.38 D-2068 PNPLA3-I148M 4.00E+11 5 −34.15 D-2069 PNPLA3-I148M 4.00E+11 5 −82.74 D-2070 PNPLA3-I148M 4.00E+11 5 −79.64 D-2071 PNPLA3-I148M 4.00E+11 5 −48.80 D-2071 PNPLA3-WT 4.00E+11 5 −59.43 D-2075 PNPLA3-I148M 4.00E+11 5 −79.10 D-2075 PNPLA3-WT 4.00E+11 5 −60.60 D-2079 PNPLA3-I148M 4.00E+11 5 −50.08 D-2072 PNPLA3-I148M 4.00E+11 5 −44.40 D-2072 PNPLA3-WT 4.00E+11 5 −43.75 D-2077 PNPLA3-I148M 4.00E+11 5 −28.46 D-2076 PNPLA3-I148M 4.00E+11 5 −83.25 D-2078 PNPLA3-I148M 4.00E+11 5 −49.10 D-2078 PNPLA3-WT 4.00E+11 5 −3.45 D-2073 PNPLA3-I148M 4.00E+11 5 −39.42 D-2074 PNPLA3-I148M 4.00E+11 5 −46.68 D-2074 PNPLA3-WT 4.00E+11 5 −5.11 D-2084 PNPLA3-I148M 4.00E+11 5 −87.49 D-2084 PNPLA3-I148M 8.00E+11 5 −88.75 D-2084 PNPLA3-I148M 8.00E+11 1 −16.49 D-2084 PNPLA3-WT 8.00E+11 1 −15.42 D-2085 PNPLA3-I148M 4.00E+11 5 −77.14 D-2085 PNPLA3-I148M 8.00E+11 5 −84.42 D-2085 PNPLA3-I148M 8.00E+11 1 −25.19 D-2085 PNPLA3-WT 8.00E+11 1 −21.14 D-2086 PNPLA3-I148M 4.00E+11 5 −64.54 D-2086 PNPLA3-I148M 4.00E+11 5 −52.95 D-2087 PNPLA3-I148M 4.00E+11 5 −20.15 D-2088 PNPLA3-I148M 4.00E+11 5 −47.18 D-2088 PNPLA3-I148M 8.00E+11 5 −66.96 D-2089 PNPLA3-I148M 8.00E+11 5 −85.47 D-2089 PNPLA3-WT 8.00E+11 1 −21.01 D-2089 PNPLA3-I148M 8.00E+11 1 −34.21 D-2089 PNPLA3-I148M 1.00E+12 5 −90.55 D-2090 PNPLA3-I148M 8.00E+11 5 −89.58 D-2090 PNPLA3-I148M 8.00E+11 1 −50.13 D-2090 PNPLA3-WT 8.00E+11 1 8.76 D-2091 PNPLA3-WT 8.00E+11 5 −35.70 D-2092 PNPLA3-I148M 8.00E+11 5 −92.34 D-2092 PNPLA3-I148M 8.00E+11 1 −51.35 D-2092 PNPLA3-WT 8.00E+11 1 −42.88 D-2093 PNPLA3-I148M 8.00E+11 5 −83.87 D-2094 PNPLA3-I148M 8.00E+11 5 −70.12 D-2095 PNPLA3-I148M 8.00E+11 1 −29.95 D-2095 PNPLA3-WT 8.00E+11 1 67.40 D-2095 PNPLA3-I148M 8.00E+11 5 −85.42 D-2096 PNPLA3-WT 8.00E+11 5 −90.44 D-2081 PNPLA3-I148M 8.00E+11 5 6.25 D-2081 PNPLA3-I148M 8.00E+11 5 −11.81 D-2097 PNPLA3-I148M 8.00E+11 5 −87.61 D-2098 PNPLA3-I148M 8.00E+11 5 −79.84 D-2099 PNPLA3-I148M 8.00E+11 5 −84.36 D-2100 PNPLA3-I148M 8.00E+11 5 −79.67 D-2101 PNPLA3-I148M 8.00E+11 5 −89.64 D-2102 PNPLA3-I148M 8.00E+11 5 −61.49 D-2103 PNPLA3-I148M 8.00E+11 5 −19.65 D-2104 PNPLA3-I148M 8.00E+11 5 −79.70 D-2104 PNPLA3-I148M 1.00E+12 5 −82.87 D-2105 PNPLA3-I148M 8.00E+11 5 −84.49 D-2105 PNPLA3-I148M 1.00E+12 5 −87.71 D-2152 PNPLA3-I148M 1.00E+12 5 −39.35 D-2153 PNPLA3-I148M 1.00E+12 5 −79.04 D-2154 PNPLA3-I148M 1.00E+12 5 −66.72 D-2155 PNPLA3-I148M 1.00E+12 5 −44.68 D-2156 PNPLA3-I148M 1.00E+12 5 −84.72 D-2157 PNPLA3-I148M 1.00E+12 5 −17.25 D-2280 PNPLA3-I148M DM 1.00E+12 5 −99.70 D-2280 PNPLA3 WT 1.00E+12 5 −51.13 D-2295 PNPLA3-WT 1.00E+12 5 −35.90 D-2295 PNPLA3-I148M DM 1.00E+12 5 −94.68 D-2296 PNPLA3-WT 1.00E+12 5 −23.24 D-2296 PNPLA3-I148M DM 1.00E+12 5 −92.78 D-2297 PNPLA3-WT 1.00E+12 5 43.71 D-2297 PNPLA3-I148M DM 1.00E+12 5 −94.59 D-2324 PNPLA3-WT 1.00E+12 5 6.53 D-2324 PNPLA3-I148M DM 1.00E+12 5 −97.39 D-2326 PNPLA3-WT 1.00E+12 5 −8.25 D-2326 PNPLA3-I148M DM 1.00E+12 5 −77.82 D-2328 PNPLA3-WT 1.00E+12 5 −2.12 D-2328 PNPLA3-I148M DM 1.00E+12 5 −92.49

TABLE 9 Day 15 PNPLA3 knockdown assay AAV Dose PNPLA3 Duplex particles/ administered knockdown number AAV vector animal (mg/kg) (%) D-2092 PNPLA3-I148M 1.00E+12 3 −87.58 D-2092 PNPLA3-I148M 1.00E+12 5 −93.96 D-2095 PNPLA3-I148M 1.00E+12 3 −81.73 D-2095 PNPLA3-I148M 1.00E+12 5 −72.99 D-2131 PNPLA3-I148M 1.00E+12 5 −66.02 D-2186 PNPLA3-I148M 1.00E+12 5 −87.67 D-2089 PNPLA3-I148M 1.00E+12 5 −95.01 D-2104 PNPLA3-I148M 1.00E+12 5 −76.01 D-2105 PNPLA3-I148M 1.00E+12 5 −72.67 D-2123 PNPLA3-I148M 1.00E+12 5 −56.93 D-2128 PNPLA3-I148M 1.00E+12 5 −37.26 D-2138 PNPLA3-I148M 1.00E+12 5 −62.16 D-2149 PNPLA3-I148M 1.00E+12 5 −72.34 D-2156 PNPLA3-I148M 1.00E+12 5 −75.81 D-2259 PNPLA3-I148M 1.00E+12 5 −79.15 D-2260 PNPLA3-I148M 1.00E+12 5 −69.97 D-2261 PNPLA3-I148M 1.00E+12 5 −50.40 D-2262 PNPLA3-I148M 1.00E+12 5 −84.36 D-2263 PNPLA3-I148M 1.00E+12 5 −77.08 D-2264 PNPLA3-I148M 1.00E+12 5 −40.86 D-2269 PNPLA3-I148M 1.00E+12 5 −37.55 D-2270 PNPLA3-I148M 1.00E+12 5 −77.42 D-2271 PNPLA3-I148M 1.00E+12 5 −26.87 D-2272 PNPLA3-I148M 1.00E+12 5 −56.05 D-2280 PNPLA3-I148M DM 1.00E+12 3 −86.30 D-2287 PNPLA3-I148M DM 1.00E+12 3 −94.73 D-2289 PNPLA3-I148M DM 1.00E+12 3 −93.48 D-2292 PNPLA3-I148M DM 1.00E+12 3 −82.48 D-2297 PNPLA3-I148M DM 1.00E+12 3 −72.61 D-2322 PNPLA3-I148M DM 1.00E+12 3 −32.92 D-2324 PNPLA3-I148M DM 1.00E+12 3 −87.56 D-2327 PNPLA3-I148M DM 1.00E+12 3 −86.70 D-2345 PNPLA3-WT 1.00E+12 5 −83.48 D-2346 PNPLA3-WT 1.00E+12 5 −75.93 D-2347 PNPLA3-WT 1.00E+12 5 −22.83 D-2348 PNPLA3-WT 1.00E+12 5 −20.09 D-2349 PNPLA3-WT 1.00E+12 5 −67.70 D-2350 PNPLA3-WT 1.00E+12 5 −57.51 D-2351 PNPLA3-WT 1.00E+12 5 −56.11 D-2352 PNPLA3-I148M DM 1.00E+12 3 −92.21 D-2353 PNPLA3-I148M DM 1.00E+12 3 −89.55 D-2354 PNPLA3-I148M DM 1.00E+12 3 −90.21 D-2358 PNPLA3-I148M DM 1.00E+12 3 −95.10 D-2359 PNPLA3-I148M DM 1.00E+12 3 −91.17 D-2360 PNPLA3-I148M DM 1.00E+12 3 −63.98 D-2361 PNPLA3-I148M DM 1.00E+12 3 −92.47 D-2362 PNPLA3-I148M DM 1.00E+12 3 −95.10 D-2364 PNPLA3-I148M DM 1.00E+12 3 −95.31 D-2370 PNPLA3-I148M DM 1.00E+12 3 −95.99 D-2370 PNPLA3 WT 1.00E+12 3 11.88 D-2371 PNPLA3-I148M DM 1.00E+12 3 −91.14 D-2372 PNPLA3-I148M DM 1.00E+12 3 −93.71 D-2373 PNPLA3-I148M DM 1.00E+12 3 −73.80 D-2374 PNPLA3-I148M DM 1.00E+12 3 −75.98 D-2375 PNPLA3-I148M DM 1.00E+12 3 −96.68 D-2376 PNPLA3-I148M DM 1.00E+12 3 −96.78 D-2377 PNPLA3-I148M DM 1.00E+12 3 −96.88 D-2378 PNPLA3-I148M DM 1.00E+12 3 −97.60 D-2379 PNPLA3-I148M DM 1.00E+12 3 −94.73 D-2380 PNPLA3-I148M DM 1.00E+12 3 −94.73 D-2381 PNPLA3-I148M DM 1.00E+12 3 −76.24 D-2382 PNPLA3-I148M DM 1.00E+12 3 −80.33 D-2383 PNPLA3-I148M DM 1.00E+12 3 −71.98 D-2384 PNPLA3-I148M DM 1.00E+12 3 −87.24 D-2385 PNPLA3-I148M DM 1.00E+12 3 −78.77 D-2386 PNPLA3-I148M DM 1.00E+12 3 −70.58 D-2387 PNPLA3-I148M DM 1.00E+12 3 −67.09 D-2390 PNPLA3-I148M DM 1.00E+12 3 −92.97 D-2391 PNPLA3-I148M DM 1.00E+12 3 −94.10 D-2392 PNPLA3-I148M DM 1.00E+12 3 −92.14 D-2395 PNPLA3-I148M DM 1.00E+12 3 −85.63 D-2396 PNPLA3-I148M DM 1.00E+12 3 −94.63 D-2397 PNPLA3-I148M DM 1.00E+12 3 −92.90 D-2398 PNPLA3-I148M DM 1.00E+12 3 −95.48 D-2399 PNPLA3-I148M DM 1.00E+12 3 −93.40 D-2400 PNPLA3-I148M DM 1.00E+12 3 −97.55 D-2401 PNPLA3-I148M DM 1.00E+12 3 −96.98 D-2402 PNPLA3-I148M DM 1.00E+12 3 −97.25 D-2403 PNPLA3 WT 1.00E+12 3 36.90 D-2404 PNPLA3-I148M DM 1.00E+12 3 −95.08 D-2405 PNPLA3-I148M DM 1.00E+12 3 −96.93 D-2406 PNPLA3 WT 1.00E+12 3 28.80 D-2395 PNPLA3-I148M DM 1.00E+12 3 −17.25 D-2396 PNPLA3-I148M DM 1.00E+12 3 −93.70 D-2413 PNPLA3-I148M DM 1.00E+12 3 −94.80 D-2415 PNPLA3-I148M DM 1.00E+12 3 −90.95 D-2418 PNPLA3-I148M DM 1.00E+12 3 −88.45 D-2419 PNPLA3 WT 1.00E+12 3 43.72 D-2453 PNPLA3 WT 1.00E+12 3 −81.33 D-2454 PNPLA3 WT 1.00E+12 3 −83.38 D-2455 PNPLA3 WT 1.00E+12 3 −68.85 D-2456 PNPLA3 WT 1.00E+12 3 −89.03 D-2460 PNPLA3 WT 1.00E+12 3 −68.65 D-2461 PNPLA3 WT 1.00E+12 3 −47.85

TABLE 10 Day 22 PNPLA3 knockdown assay AAV Dose PNPLA3 Duplex particles/ administered knockdown number AAV vector animal (mg/kg) (%) D-2092 PNPLA3-I148M 1.00E+12 3 −74.61 D-2092 PNPLA3-I148M 1.00E+12 5 −85.78 D-2095 PNPLA3-I148M 1.00E+12 3 −27.78 D-2095 PNPLA3-I148M 1.00E+12 5 −33.60 D-2324 PNPLA3-I148M DM 1.00E+12 3 −56.68 D-2324 PNPLA3-I148M DM 1.00E+12 5 −88.02 D-2089 PNPLA3-I148M 1.00E+12 5 −80.86 D-2104 PNPLA3-I148M 1.00E+12 5 −65.61 D-2105 PNPLA3-I148M 1.00E+12 5 −39.67 D-2280 PNPLA3-I148M DM 1.00E+12 5 −84.12 D-2297 PNPLA3-I148M DM 1.00E+12 3 −58.01 D-2297 PNPLA3-I148M DM 1.00E+12 5 −76.43 D-2352 PNPLA3-I148M DM 1.00E+12 5 −92.74 D-2353 PNPLA3-I148M DM 1.00E+12 5 −84.54 D-2354 PNPLA3-I148M DM 1.00E+12 5 −89.06 D-2355 PNPLA3-I148M DM 1.00E+12 5 −95.53 D-2356 PNPLA3-I148M DM 1.00E+12 5 −94.99 D-2357 PNPLA3-I148M DM 1.00E+12 5 −97.37

TABLE 11 Day 28 PNPLA3 knockdown assay AAV Dose PNP Duplex particles/ administered knockdown number AAV vector animal (mg/kg) (%) D-2092 PNPLA3-I148M 1.00E+12 3 −72.57 D-2092 PNPLA3-I148M 1.00E+12 5 −82.74 D-2095 PNPLA3-I148M 1.00E+12 3 −32.93 D-2095 PNPLA3-I148M 1.00E+12 5 −55.31 D-2089 PNPLA3-I148M 1.00E+12 5 −63.49 D-2104 PNPLA3-I148M 1.00E+12 5 −44.39 D-2105 PNPLA3-I148M 1.00E+12 5 −39.80 D-2370 PNPLA3-I148M DM 1.00E+12 3 −89.28 D-2400 PNPLA3-I148M DM 1.00E+12 3 −88.23 D-2401 PNPLA3-I148M DM 1.00E+12 3 −91.95 D-2402 PNPLA3-I148M DM 1.00E+12 0.5 −50.65 D-2402 PNPLA3-I148M DM 1.00E+12 1 −69.37 D-2402 PNPLA3-I148M DM 1.00E+12 3 −96.43 D-2402 PNPLA3-I148M DM 1.00E+12 3 −85.44 D-2402 PNPLA3-I148M DM 1.00E+12 3 −90.52 D-2404 PNPLA3-I148M DM 1.00E+12 3 −95.67 D-2419 PNPLA3-I148M DM 1.00E+12 0.5 −68.83 D-2419 PNPLA3-I148M DM 1.00E+12 1 −76.88 D-2419 PNPLA3-I148M DM 1.00E+12 3 −97.53 D-2419 PNPLA3-I148M DM 1.00E+12 3 −93.95 D-2419 PNPLA3-I148M DM 1.00E+12 3 −89.66 D-2419 PNPLA3-I148M DM 1.00E+12 3 −93.25 D-2420 PNPLA3-I148M DM 1.00E+12 3 −95.93 D-2421 PNPLA3-I148M DM 1.00E+12 0.5 −50.01 D-2421 PNPLA3-I148M DM 1.00E+12 1 −66.30 D-2421 PNPLA3-I148M DM 1.00E+12 3 −94.99 D-2421 PNPLA3-I148M DM 1.00E+12 3 −80.05 D-2421 PNPLA3 WT 1.00E+12 3 −36.65 D-2421 PNPLA3-I148M DM 1.00E+12 3 −91.08 D-2425 PNPLA3-I148M DM 1.00E+12 3 −16.07 D-2426 PNPLA3-I148M DM 1.00E+12 3 −37.54 D-2427 PNPLA3-I148M DM 1.00E+12 3 −25.19 D-2428 PNPLA3-I148M DM 1.00E+12 3 −16.71 D-2437 PNPLA3-I148M DM 1.00E+12 3 −93.78 D-2438 PNPLA3-I148M DM 1.00E+12 3 −90.63 D-2439 PNPLA3-I148M DM 1.00E+12 3 −88.10 D-2440 PNPLA3-I148M DM 1.00E+12 3 −95.25 D-2441 PNPLA3-I148M DM 1.00E+12 3 −90.13 D-2442 PNPLA3-I148M DM 1.00E+12 3 −57.24 D-2443 PNPLA3-I148M DM 1.00E+12 3 −95.43 D-2444 PNPLA3-I148M DM 1.00E+12 3 −90.58 D-2445 PNPLA3-I148M DM 1.00E+12 3 −84.55 D-2446 PNPLA3-I148M DM 1.00E+12 3 −81.50 D-2427 PNPLA3-I148M DM 1.00E+12 3 −90.09 D-2462 PNPLA3-I148M DM 1.00E+12 3 −89.20 D-2463 PNPLA3-I148M DM 1.00E+12 3 −41.25 D-2464 PNPLA3-I148M DM 1.00E+12 3 −60.53 D-2465 PNPLA3-I148M DM 1.00E+12 3 −91.35 D-2466 PNPLA3-I148M DM 1.00E+12 3 −93.68 D-2467 PNPLA3-I148M DM 1.00E+12 3 −85.15 D-2472 PNPLA3-I148M DM 1.00E+12 0.5 −46.58 D-2472 PNPLA3-I148M DM 1.00E+12 1 −74.47 D-2472 PNPLA3-I148M DM 1.00E+12 3 −88.79 D-2472 PNPLA3 WT 1.00E+12 3 −23.16 D-2472 PNPLA3-I148M DM 1.00E+12 3 −90.54 D-2473 PNPLA3-I148M DM 1.00E+12 0.5 −57.95 D-2473 PNPLA3-I148M DM 1.00E+12 1 −71.96 D-2473 PNPLA3-I148M DM 1.00E+12 3 −91.70 D-2473 PNPLA3-I148M DM 1.00E+12 3 −85.94 D-2473 PNPLA3 WT 1.00E+12 3 −18.70

TABLE 12 Day 42 PNPLA3 knockdown assay AAV Dose PNPLA3 Duplex particles/ administered knockdown number AAV vector animal (mg/kg) (%) D-2402 PNPLA3-1148M DM 1.00E+12 1 −57.74 D-2402 PNPLA3-1148M DM 1.00E+12 3 −83.75 D-2419 PNPLA3-1148M DM 1.00E+12 1 −71.07 D-2419 PNPLA3-1148M DM 1.00E+12 3 −70.8  D-2421 PNPLA3-1148M DM 1.00E+12 1 −62.21 D-2421 PNPLA3-1148M DM 1.00E+12 3 −80.12 D-2472 PNPLA3-1148M DM 1.00E+12 1 −60.54 D-2472 PNPLA3-1148M DM 1.00E+12 3 −74.77 D-2473 PNPLA3-1148M DM 1.00E+12 1 −54.55 D-2473 PNPLA3-1148M DM 1.00E+12 3 −81.13

Example 5: Prevention and Rescue of NAFLD by siRNA Molecules in a Humanized PNPLA3^(rs738409-rs738408) Mouse Model

The ‘American Lifestyle-Induced Obesity Syndrome’, or ALIOS mouse model for NAFLD/NASH is developed by feeding mice with a diet high in trans-fats (45% of the total amount of fat) and sugar (Tetri 2008). For these studies, eight to ten-week old C57BL/6NCrl male mice (Charles River Laboratories Inc.) were injected with AAV-empty vector or AAV8-PNPLA3^(rs738409-rs738408), as described previously. At the time of AAV injection, mice were either maintained on a normal chow diet or placed on the ALIOS diet (Envigo, TD.06303) complete with drinking water composed of 55% fructose and 45% glucose (Sigma, F0127 and G7021, respectively) until harvest. In previous experiments, it was established that over-expression of PNPLA3^(rs738409-rs738408), in this context, both accelerates and worsens NAFLD phenotypes (data not shown).

Two weeks after AAV injection and diet initiation, mice were treated with a single dose of siRNA D-2324 (0.5 mM), via subcutaneous injection, at 5.0 milligrams per kilogram of animal, diluted in phosphate buffered saline (Thermo Fisher Scientific, 14190-136) or a vehicle control. Dosing was repeated biweekly until harvest. At the time of harvest, body weights were collected, followed by serum via cardiac puncture under isoflurane anesthesia, followed by liver weights. The median lobe was fixed via 10% neutral buffered formalin, followed by paraffin processing and embedding. The remainder of the liver was snap frozen for content and gene expression analysis, as described previously.

Snap frozen liver tissue was processed for RNA and gene expression analysis, as described previously. Results are shown as both the raw Ct value and relative mRNA expression of the indicated gene normalized to mouse Gapdh. (TaqMan™ assays from Invitrogen: human PNPLA3, hs00228747_m1; mouse Pnpla3, Mm00504420_m1; mouse Gapdh, 4352932E). Formalin-fixed tissues were processed for Hemotoxylin and Eosin staining (Dako, CS70030-2, CS70130-2, respectively) according to the manufacturer's instructions. Scoring for steatosis and inflammation was performed by a board-certified pathologist.

Serum analysis included TIMP1, a biomarker associated with NASH and NASH-related fibrosis (Youssani 2011). The TIMP1 ELISA (R&D Systems, MTM100) was performed according to the manufacturer's instructions.

FIG. 2A-G. To evaluate the ability of a PNPLA3^(rs738409-rs738408)-specific siRNA molecule D-2324 to prevent the development of phenotypes associated with NAFLD and overexpression of PNPLA3^(rs738409-rs738408), mice received AAV8-empty vector (EV), or AAV8-PNPLA3^(rs738409-rs738408), or vehicle, and were maintained on a regular chow diet or transitioned to the ALIOS diet. Two weeks after AAV injections, mice were treated with siRNA or vehicle, every other week for six weeks; a total of three injection rounds. Mice were harvested at the eight-week time point. Results are presented as the group average and standard error, N=8 per group. Asterisks represent statistical significance to the AAV8-PNPLA3^(rs738409-rs738408) cohort, generated by One-way ANOVA employing Dunnett's multiple comparisons test. (A) The ratio of liver weight (grams) to body weight (grams) at the time of harvest. Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, **=0.0018, AAV-PNPLA3^(rs738409-rs738408)+siRNA, ****<0.0001. (B) Confirmation of human PNPLA3 mRNA expression and silencing in the liver by qPCR. (left) Raw Ct value and (right) relative fold mRNA expression normalized to mouse Gapdh; comparing the ALIOS-fed PNPLA3^(rs738409-rs738408)+vehicle and PNPLA3^(rs738409-rs738408)+siRNA groups. (C) Analysis of mouse Pnpla3 mRNA expression in liver by qPCR indicates endogenous Pnpla3 is not significantly altered by AAV-mediated over-expression or siRNA silencing. (left) Raw Ct value and (right) relative fold mRNA expression normalized to mouse Gapdh; comparing the chow-fed no-AAV group to the ALIOS-fed PNPLA3^(rs738409-rs738408)+vehicle and PNPLA3^(rs738409-rs738408)+siRNA groups. (D) Hepatic triglyceride content presented as milligrams of triglyceride per gram of liver tissue. Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, *=0.0393, AAV-PNPLA3^(rs738409-rs738408) siRNA, **=0.0063. (E) Serum TIMP1 presented as picograms per milliliter of serum. Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, ****<0.0001, AAV-PNPLA3^(rs738409-rs738408)+siRNA, ****<0.0001. (F) Histological indication of steatosis based on H&E staining, scored as: Within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, no significance, AAV-PNPLA3^(rs738409-rs738408)+siRNA, **=0.0012. (G) Histological indication of inflammation based on H&E staining, scored as: Within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, ****<0.0001, AAV-PNPLA3^(rs738409-rs738408)+siRNA, ****<0.0001.

FIG. 3A-G. To evaluate the ability of a PNPLA3^(rs738409-rs738408)-specific siRNA molecule to prevent further progression of PNPLA3^(rs738409-rs738408)-mediated disease after disease onset, mice received AAV8-empty vector (EV), or AAV8-PNPLA3^(rs738409-rs738408), or vehicle, and were maintained on a regular chow diet or transitioned to the ALIOS diet. Eight weeks after AAV injections and diet changes, mice were treated with siRNA or vehicle, every other week for eight more weeks; a total of four injection rounds. Mice were harvested at the sixteen-week time point. Although no change was observed in steatosis, if siRNA treatment began after disease induction, several other disease-associated end points were significantly reduced. Results are presented as averages and standard error, N=8 per group. Asterisks represent statistical significance to the AAV8-PNPLA3^(rs738409-rs738408) cohort, generated by One-way ANOVA employing Dunnett's multiple comparisons test. (A) The ratio of liver weight (grams) to body weight (grams) at the time of harvest. Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, not significant, AAV-PNPLA3^(rs738409-rs738408)+siRNA, ***=0.0006. (B) Confirmation of human PNPLA3 mRNA expression and silencing in the liver by qPCR. (left) Raw Ct value and (right) relative fold mRNA expression normalized to mouse Gapdh; comparing the ALIOS-fed PNPLA3^(rs738409-rs738408)+vehicle and PNPLA3^(rs738409-rs738408)+siRNA groups. (C) Analysis of mouse Pnpla3 mRNA expression in liver by qPCR indicates endogenous Pnpla3 is not significantly altered by AAV-mediated over-expression or siRNA silencing. (left) Raw Ct value and (right) relative fold mRNA expression normalized to mouse Gapdh; comparing the chow-fed no-AAV group to the ALIOS-fed PNPLA3^(rs738409-rs738408)+vehicle and PNPLA3^(rs738409-rs738408)+siRNA groups. (D) Hepatic triglyceride content presented as milligrams of triglyceride per gram of liver tissue. Adjusted P values: AAV-EV+vehicle, not significant, AAV-PNPLA3^(rs738409-rs738408)+siRNA, *=0.0403. (E) Serum TIMP1 presented as picograms per milliliter of serum. Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, **=0.0027, AAV-PNPLA3^(rs738409-rs738408)+siRNA, **=0.002. (F) Histological indication of steatosis based on H&E staining, scored as: Within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, not significant, AAV-PNPLA3^(rs738409-rs738408)+siRNA, not significant. (G) Histological indication of inflammation based on H&E staining, scored as: Within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, not significant, AAV-PNPLA3^(rs738409-rs738408)+siRNA, **=0.0068.

FIG. 4A-D. To evaluate the ability of a PNPLA3^(rs738409-rs738408)-specific siRNA molecule to rescue disease-associated phenotypes due to overexpression of PNPLA3^(rs738409-rs738408), liver and serum from ALIOS-fed eight-week AAV8-PNPLA3^(rs738409-rs738408)-vehicle treated mice were compared to livers and serum from ALIOS-fed sixteen-week vehicle- or siRNA-treated AAV8-PNPLA3^(rs738409-rs738408) mice. Although no change was observed in steatosis at this time point with siRNA treatment, hepatic triglyceride, serum TIMP1, and inflammation were all statistically lower at sixteen weeks compared to vehicle controls at eight weeks. Results are presented as averages and standard error, N=8 per group. Asterisks represent statistical significance to the eight-week AAV8-PNPLA3^(rs738409-rs738408) vehicle-treated cohort, generated by One-way ANOVA employing Dunnett's multiple comparisons test. (A) Hepatic triglyceride content presented as milligrams of triglyceride per gram of liver tissue. Adjusted P values: 16WK AAV-PNPLA3^(rs738409-rs738408)+vehicle, not significant; 16WK AAV-PNPLA3^(rs738409-rs738408)+siRNA, **=0.0011. (B) Serum Timp1 presented as picograms per milliliter of serum. Adjusted P values: 16WK AAV-PNPLA3^(rs738409-rs738408)+vehicle, not significant; 16WK AAV-PNPLA3^(rs738409-rs738408)+siRNA, *=0.0134. (C) Histological indication of steatosis based on H&E staining, scored as: Within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: 16WK AAV-PNPLA3^(rs738409-rs738408)+vehicle, not significant; 16WK AAV-PNPLA3^(rs738409-rs738408)+siRNA, not significant. (D) Histological indication of inflammation based on H&E staining, scored as: Within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: 16WK AAV-PNPLA3^(rs738409-rs738408)+vehicle, not significant; 16WK AAV-PNPLA3^(rs738409-rs738408)+siRNA *=0.0112.

Example 6: Prevention of Liver Fibrosis by siRNA Molecules in a Humanized PNPLA3^(rs738409-rs738408) Mouse Model

The “AMLN” diet, developed by Amylin Pharmaceuticals (Clapper 2013), is a modified version of the ALIOS diet. The feed includes a ten-fold increase in cholesterol (2%) and additional sucrose. Mice placed on the “AMLN” diet develop mild to moderate fibrosis after 20-30 weeks (Clapper, Mells and Kristiansen papers). For this study, eight to ten-week old C57BL/6NCrl male mice (Charles River Laboratories Inc.) were injected with AAV-empty vector or AAV-PNPLA3^(rs738409-rs738408), as described above, to accelerate disease onset. At the time of AAV injection, mice were continued on a normal chow diet or placed on the Envigo diet, TD.170748, complete with drinking water composed of 55% fructose and 45% glucose (Sigma, F0127 and G7021, respectively) until harvest.

Two weeks post-AAV injection and diet initiation, mice were treated with a single dose of siRNA D-2324 (0.5 mM), via subcutaneous injection, at 5.0 milligrams per kilogram of animal, diluted in phosphate buffered saline (Thermo Fisher Scientific, 14190-136) or a vehicle control. Dosing was repeated biweekly until harvest. At the time of harvest, body weights were collected, followed by serum via cardiac puncture under isoflurane anesthesia, followed by liver weights. The median lobe was fixed via 10% neutral buffered formalin, followed by paraffin processing and embedding. The remainder of the liver was snap frozen for gene expression analysis.

Snap frozen liver tissue was processed for RNA and gene expression analysis, as described previously. Results are shown as both the raw Ct value and relative mRNA expression of the indicated gene normalized to mouse Gapdh. (TaqMan™ assays from Invitrogen: human PNPLA3, hs00228747_m1; mouse Pnpla3, Mm00504420_m1; mouse Collal, Mm00801666_g1; mouse Col3a1, Mm01254471_g1; Col4a1, Mm01210125_m1; mouse Gapdh, 4352932E). Col1a1, Col3a1 and Col4a1 are extracellular matrix markers associated with hepatic stellate cell activation and liver fibrosis (Baiocchini 2016).

Formalin-fixed tissues were processed for Hemotoxylin, Eosin, and Masson's Trichrome staining (Dako, CS70030-2, CS70130-2, AR17311-2, respectively) according to the manufacturer's instructions. Anti-Smooth Muscle Actin staining was performed without antigen retrieval and using a DAKO autostainer. Slides were processed using Peroxidazed 1 and Sniper (Biocare, PX968 and BS966, respectively) and stained with monoclonal anti-Actin, alpha-Smooth Muscle antibody (Sigma, F3777), followed by Rabbit anti-FITC (Invitrogen, 711900), Envision-Rabbit HRP polymer (Dako, K4003), DAB+ (Dako, K3468), and Hemotoxylin. Scoring for the amount of steatosis, inflammation, oval cell/bile duct hyperplasia, and aSMA-positive cells was performed by a board-certified pathologist.

Serum was analyzed for mouse TIMP1 (R&D Systems, MTM100) and Mouse Cytokeratin 18-M30 (Cusabio, CSB-E14265m) according to manufacturer's instructions. In addition to TIMP1, Cytokine 18-M30 has been identified as a potential biomarker for NAFLD/NASH, including early fibrosis (Neuman 2014 and Yang 2015). FIG. 5A-L. To evaluate the ability of a PNPLA3^(rs738409-rs738408)-specific siRNA molecule to prevent the development of early fibrosis, mice received AAV8-empty vector (EV), or AAV8-PNPLA3^(rs738409-rs738408), or vehicle, and were maintained on a regular chow diet or transitioned to the AMLN diet. Two weeks after AAV injections, mice were treated with siRNA, D-2324, or vehicle, every other week for ten more weeks; a total of six injection rounds. Mice were harvested at the ten-week time point. Results are presented as averages and standard error, Chow-fed no AAV+vehicle and AMLN-fed AAV8-PNPLA3^(rs738409-rs738408)+vehicle, N=8 per group; AMLN-fed AAV8-PNPLA3^(rs738409-rs738408)+vehicle and AAV8-PNPLA3^(rs738409-rs738408)+siRNA, N=12 per group. Asterisks represent statistical significance to the AAV8-PNPLA3^(rs738409-rs738408)−vehicle-treated cohort, generated by One-way ANOVA employing Dunnett's multiple comparisons test. (A) The ratio of liver weight (grams) to body weight (grams) at the time of harvest. Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, not significant, AAV-PNPLA3^(rs738409-rs738408)+siRNA, ****_(<)0.0001. (B) Confirmation of human PNPLA3 mRNA expression and silencing in the liver by qPCR. (left) Raw Ct value and (right) relative fold mRNA expression normalized to mouse Gapdh; comparing the PNPLA3^(rs73849-rs738408)+vehicle and PNPLA3^(rs738409-rs738408)+siRNA groups. (C) Analysis of mouse Pnpla3 mRNA expression in liver by qPCR indicates endogenous Pnpla3 is not significantly altered by AAV-mediated over-expression or siRNA silencing. (left) Raw Ct value and (right) relative fold mRNA expression normalized to mouse Gapdh; comparing the chow-fed no-AAV group to the AMLN-fed PNPLA3^(rs738409-rs738408)+vehicle and PNPLA3^(rs738409-rs738408)+siRNA groups. (D) Serum Timp1 presented as picograms per milliliter of serum. Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, ****<0.0001, AAV-PNPLA3^(rs738409-rs738408)+siRNA, ****<0.0001. (E) Serum CK18m30 presented as picograms per milliliter of serum. Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, ****<0.0001, AAV-PNPLA3^(rs738409-rs738408)+siRNA, ****<0.0001. (F) Histological indication of inflammation based on H&E staining, scored as: Within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, *=0.0108, AAV-PNPLA3^(rs738409-rs738408)+siRNA, ****_(<)0.0001. (G) Histological indication of oval cell/bile duct hyperplasia based on H&E staining, scored as: Within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, not significant, AAV-PNPLA3^(rs738409-rs738408)+siRNA, **=0.0081. (H) Immunohistochemical staining for anti-Smooth Muscle Actin, scored as: Within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, *=0.0101, AAV-PNPLA3^(rs738409-rs738408)+siRNA, ***=0.0002. (I) Masson's Trichrome staining for fibrosis, scored as: Within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, not significant, AAV-PNPLA3^(rs738409-rs738408)+siRNA, not significant. (J) Mouse Collal mRNA expression in liver by qPCR. Relative fold mRNA expression normalized to mouse Gapdh. Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, ****<0.0001, AAV-PNPLA3^(rs738409-rs738408)+siRNA, ****<0.0001. (K) Mouse Col3a1 mRNA expression in liver by qPCR. Relative fold mRNA expression normalized to mouse Gapdh. Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, ****<0.0001, AAV-PNPLA3^(rs738409-rs738408)+siRNA, ****<0.0001. (L) Mouse Col4a1 mRNA expression in liver by qPCR. Relative fold mRNA expression normalized to mouse Gapdh. Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, ***<0.0005, AAV-PNPLA3^(rs738409-rs738408)+siRNA, **<0.0041. 

1. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having at least 15 contiguous nucleotides differing by no more than 3 nucleotides from an antisense sequence listed in Table 1 or 2, and wherein the RNAi construct inhibits the expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3).
 2. The RNAi construct of claim 1, wherein the antisense strand comprises a region that is complementary to a PNPLA3 mRNA sequence.
 3. The RNAi construct of claim 2, wherein the sense strand comprises a region having at least 15 contiguous nucleotides differing by no more than 3 nucleotides from an antisense sequence listed in Table 1 or
 2. 4. The RNAi construct of claim 3, wherein the construct preferentially inhibits a PNPLA3-rs738409 minor allele.
 5. (canceled)
 6. The RNAi construct of claim 3, wherein the construct preferentially inhibits a PNPLA3-rs738408 minor allele.
 7. The RNAi construct of claim 6, wherein the construct has at least 10% greater inhibition of a PNPLA3-rs738409-rs738408 double minor allele as compared to the major allele.
 8. The RNAi construct of claim 3, wherein the sense strand comprises a sequence that is sufficiently complementary to the sequence of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length. 9-11. (canceled)
 12. The RNAi construct of claim 8, wherein the sense strand and the antisense strand are each about 15 to about 30 nucleotides in length. 13-15. (canceled)
 16. The RNAi construct of claim 8, wherein the RNAi construct comprises at least one blunt end.
 17. The RNAi construct of claim 16, wherein the RNAi construct comprises at least one nucleotide overhang of 1 to 4 unpaired nucleotides.
 18. (canceled)
 19. The RNAi construct of claim 17, wherein the RNAi construct comprises a nucleotide overhang at the 3′ end of the sense strand, the 3′ end of the antisense strand, or the 3′ end of both the sense strand and the antisense strand.
 20. The RNAi construct of claim 17, wherein the nucleotide overhang comprises a 5′-UU-3′ dinucleotide or a 5′-dTdT-3′ dinucleotide.
 21. The RNAi construct of claim 1, wherein the RNAi construct comprises at least one modified nucleotide, wherein the modified nucleotide is a 2′-modified nucleotide.
 22. (canceled)
 23. The RNAi construct of claim 21, wherein the modified nucleotide is a 2′-fluoro modified nucleotide, a 2′-O-methyl modified nucleotide, a 2′-O-methoxyethyl modified nucleotide, a 2′-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), a glycol nucleic acid, an inverted base or combinations thereof. 24-26. (canceled)
 27. The RNAi construct of claim 1, wherein the RNAi construct comprises at least one phosphorothioate internucleotide linkage. 28-32. (canceled)
 33. The RNAi construct of claim 1, wherein the RNAi construct reduces the expression level of PNPLA3 in liver cells following incubation with the RNAi construct as compared to the PNPLA3 expression level in liver cells that have been incubated with a control RNAi construct.
 34. The RNAi construct of claim 27, wherein the liver cells are Hep3B or HepG2 cells.
 35. The RNAi construct of claim 1, wherein the RNAi construct inhibits at least 10% of PNPLA3 expression at 5 nM in Hep3B cells in vitro, inhibits at least 10% of PNPLA3 expression at 5 nM in HepG2 cells in vitro, inhibits PNPLA3 expression in Hep3B cells with an IC50 of less than about 1 nM, or inhibits PNPLA3 expression in HepG2 cells with an IC50 of less than about 1 nM. 36-38. (canceled)
 39. A pharmaceutical composition comprising the RNAi construct of claim 1 and a pharmaceutically acceptable carrier, excipient, or diluent.
 40. A method for reducing the expression of PNPLA3 in a patient in need thereof comprising administering to the patient the RNAi construct of claim
 1. 41. The method of claim 40, wherein the expression level of PNPLA3 in hepatocytes is reduced in the patient following administration of the RNAi construct as compared to the PNPLA3 expression level in a patient not receiving the RNAi construct. 